UABF

URINE

Handling and Preservation

▪ Examine within 1 hr if kept at RT

▪ Delay in Examination: REFRIGERATE.; must be examined within 2 – 4 hrs
Methods of Collection
▪ Midstream or Clean catch
Used when a urinary tract is suspected.
Collect the mid-portion of the void. (it minimizes bacterial contamination
from the skin and genital region)
▪ Random or Non-clean catch
Used if bacteriological testing is not indicated
No special instructions or preparations
▪ Pediatric Collection Bag
Used for infants and small children
Caregiver applies the bag over the genital region after cleansing the area
▪ Catheter
Used when the patient cannot void on their own.
A thin tube is inserted in to the urethra up to the bladder, allowing the
urine to flow through into the collection bag
▪ Suprapubic Aspiration
Used for patients with urinary obstruction or to obtain a sterile sample
from the patient usually infants and children.
Clinician inserts a needle into the bladder and aspirates the urine into a
sterile syringe.
Time of Collection
▪ Random or routine collection – performed when timing is not important
▪ First morning collection – performed to obtain a concentrated specimen.
Patient collects their first void after waking up
▪ Timed Collection
-performed to obtain sample during a set of time frame (e.g., 2 hrs or 24
hrs)
-patient collects all voids during the specified time and TOTAL VOLUME is
noted.
PHYSICAL examination
Normal fresh urine: pale-yellow to dark-yellow and clear sage review center

Pink Red/Red/Red Hb, blood(fresh/old), methemoglobin, porphyrins, rifampin, L-dopa

brown or beets
Amber or brown bilirubin

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 Green or blue green Biliverdin, medications containing a dye, or infection with
pseudomonas
Bright orange Pyridium
Dark-brown or black Homogentisic acid (alkaptonuria) or melanin
Cloudy and turbid May contain formed or cellular elements (e.g., crystals, bacteria, cells

Odor
Normal fresh urine has a mild odor
Sulfur odor Asparagus metabolites
Musty or mousy Phenylketonuria and some liver disease
ammonia Old samples or samples associated with UTI
Sweet smell MSUD – Maple Syrup Urine Disease
Pungent odor ketones

CHEMICAL examination

Dipstick Procedure
▪ Mix urine by inversion
▪ Moisten strip completely but briefly
▪ Tap off excess urine and begin timing
▪ Compare the color of each test pad with the color blocks on the dipstick
container at specified times
▪ Record results
Semiquantitative
Percentage concentration as in mg/dl

Specific Gravity
- density of urine is compared to the density of water
Correlates with urine osmolality
Not always correlated with darker color or turbidity

Methods of Analysis

Refractometer
INDIRECT MEASUREMENT based on the refractive index of the light.
Affected by high concentrations of protein, glucose, radiographic contrast
media sage review center

Dipstick
Color reaction from deep blue green to yellow-green based on the pKa
change of polyelectrolytes in relation to solutions ionic concentration

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 Reference interval: 1.003 – 1.040
Not affected by the presence of radiocontrast dyes or high
concentrations of protein or glucose.

pH
▪ Dipstick reaction based on color change from orange (acid) to blue (alkaline) due
to the DOUBLE INDICATOR SYSTEM.
▪ Urine becomes alkaline on standing because of bacteria that produce urease
▪ Urinary pH affected by acid-base disorders
▪ Fixed urine pH >6.5 may indicate renal disorders such as renal tubular acidosis

SQ: Historically used as a screening test for inborn error of metabolism such as galactosemia.
a. Ictotest
b. Acetest
c. Dipstick
d. Clinitest,

SQ: Sources of false positive for glucose dipstick includes the following except:
a. Oxidizing agent
b. Peroxide
c. Bleach
d. Ascorbic acid,
Glucose
Dipstick
▪ Color reaction generally from blue to green to brown based on a double
sequential enzyme reaction.
▪ Manufacturers may vary in dye used and color may turn from pale yellow to
darker green.
▪ Specific for glucose
▪ Detects at 100 mg/dl
▪ Reference Interval: negative
▪ False positive: due to contamination with peroxide, bleach, or other strong
oxidizer.
▪ False negative due to the presence of reducing substances such as ascorbic acid
and salicylates sage review center
▪ Predominant method for screening glucosuria in pediatric and adult patients.

Clinitest tablets
▪ Copper reduction test
▪ Color change from blue to green to orange due to presence and amount of
reducing substances
▪ Specific for reducing substances e.g., galactose, lactose, ascorbic acid, and
homogentisic acid

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 ▪ Detects at 250 mg/dl
▪ Reference Interval: negative
▪ Historically used as screening test for inborn errors of metabolism in newborns
and children (e.g., galactosemia)

SQ: The ketone detected in urine dipstick:

a. Acetoacetic acid, b. acetone d. beta-hydroxybutyric acid d. all of the above

Ketones
• Ketone bodies (acetoacetic acid, acetone, and Beta-hydroxybutyric acid) are by
products of excess acetyl-CoA production in fat catabolism.
• Dipstick
o Purple color of increasing intensity when acetoacetic acid reacts
with sodium nitroprusside
o Specific for acetoacetic acid
o Detects at 5 – 10 mg/dl
o Reference Interval: negative
o False positive occurs in highly pigmented urine or in presence of
levodopa metabolites or sulfhydryl compounds
• Acetest tablets
o Development of lavender to purple color when urinary ketone
reacts with Na nitroprusside in the tablet
o Lactose and disodium phosphate in tablet enhances the reaction
o Specific for acetoacetic acid and acetone
o detects at approximately 5 – 10 mg/dl
o Reference interval: negative

Protein
❖ Dipstick
• Reaction is based on protein error of indicators using tetrabromphenol
blue buffered to pH3
• Color changes from yellow green to blue green as more protein is
detected

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 • Detects at 15 – 30 mg/dl
• Reference Interval: negative
❖ Screening test for proteinuria due to infections or kidney disease such as
nephritis
❖ Transitory or intermittent benign proteinuria seen after stress or exercise
❖ Early stages of diabetic nephropathy may not yield a positive reaction;
microalbumin strips available
❖ Nephrotic syndrome associated with significant proteinuria

Occult Blood
• Means hidden blood: not obvious by direct observation
• Dipstick
o Test pad contains chromagen and a peroxide, which reacts with
pseudo peroxidase activity of the heme moiety to produce a color
change.
o Greenish blue color develops in the presence of RBCs, hemoglobin
or myoglobin
o More sensitive to myoglobin than intact rbcs
o Detects 5-10 rbcs/uL or hb @ 0.015 – 0.062 mg/dl
o Small numbers of rbcs produce a spotted reaction
o Reference interval is negative
o False positives due to presence of oxidizing agents (eg, bleach),
povidone-iodine, and some bacterial infections.
o False positives due to presence of ascorbic acid, captopril,
formalin, and high levels of protein and or nitrites
• Associated with hematuria, hemoglobinuria, myoglobinuria

Bilirubin
• By-products of heme catabolism. Conjugated form is water soluble and usually found in
urine sagereviewcenter
• Dipstick
o Color change from tan to purple based on reactions of diazonium salts with
bilirubin
o Specific only for conjugated bilirubin; unconjugated bilirubin is not found in urine
o Detects 0.2 – 0.4 mg/dl
o False positive is due to presence of indican, chlorpromazine, and pyridine
o False negative due to the presence of ascorbic acid and exposure to light
o Bilirubinuria is seen in liver disease and conjugated hyperbilirubinemia
• Ictotest tablet

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 o Development of blue or purple color on test mat following reaction of di
azo salts and urinary bilirubin
o More sensitive than dipstick method
o Used as a confirmatory test for positive dipstick results
o Reference interval: negative

Urobilinogen
• Colorless by-product of conjugated bilirubin hydrolysis by intestinal bacteria
• Dipstick
o Color change from light pink to red based on the reaction of urobilinogen
with Ehrlich reagent
o Detects at 0.2 mg/dl
o Reference interval: <1.0 mg/dl
o False positives due to presence of salicylates, pyridine, and sulfonamides
o False negative due to presence of formalin, high concentration of nitrites,
or exposure to light
o Urobilinogen is increased in liver disease
o Not detectable in biliary obstruction
Nitrites
• Detects presence of nitrate-reducing bacteria in the urine such as Escherichia coli
• Dipstick
o Color change from white to pink based on reaction of nitrite with
p-arsanilic acid to form a diazonium product which then reacts
with quinoline
o Detects at 0.06 - 0.10 mg/dl

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 o Reference interval: negative
o False positive due to the presence of pyridine; may also occur if
the sample is not tested in a timely fashion or allowed to sit at
room temperature
o False negatives due to high specific gravity, presence of ascorbic
acid or high levels of urobilinogen
o Detects esterase from >10 leukocytes/ml
o Reference interval is negative
• Not all bacteria reduce nitrate to nitrite
• Poor correlation between the nitrite test and positive cultures or random
samples
• Best to use first morning sample since nitrites form after 4-hour incubation in
the bladder

Leukocyte Esterase
• Dipstick
o Color develops as a result of hydrolysis reaction of esters and diazonium salt
catalyzed by esterases from granulocytic leukocytes
o False positive due to the presence of strong oxidizing agents (e.g., bleach),
formalin, vaginal fluid, eosinophil, and trichomonas
o False negatives due to high specific gravity, presence of ascorbic acid, some
antibiotics, and high concentrations of protein or glucose
o Detects esterase from >10 leukocytes/ml
o Reference interval is negative
▪ Screening test for WBCs, particularly neutrophils

MICROSCOPIC EXAMINATION
General Considerations
1) The results of microscopic should correlate with the physical and chemical test
results
2) Contamination is common, especially in voided specimens when no effort is made to
obtain a “clean catch” specimen
3) Cellular elements tend to lyse in dilute (hypotonic) or alkaline urines
4) Reference intervals vary due to variation in methods used to concentrate the
sediment by centrifugation (e.g., sample volume, speed, time)
5) Unpreserved urine results in cell degradation, bacterial proliferation, glycolysis, pH
changes and if exposed to light, decreases in bilirubin and urobilinogen
Sediment Preparation
1) Centrifuge 12 ml of well mixed urine (1500 – 2000 rpm) for 5 minutes
2) Suction or pour off all but 1 ml of urine
3) Resuspend sediment in remaining 1 mL and place 50 Ul on a glass slide; add
coverslip

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 4) Examine under low power with dimmed light or with phase contrast microscopy for
cast and crystals; report as number per lpf.
5) Examine under high dry power for rbcs, wbcs, bacteria, yeast, and epithelial cells,;
report as number per hpf

Epithelial cells

▪ Squamous epithelial cells

o Large (30 – 50 um), flagstone-shaped cells with small central nuclei

o Appear flat with abundant cytoplasm
o Originate from the superficial lining (skin cells) of the urethra and vagina
o Contaminant commonly seen in non-clean catch specimens; significant
numbers may be associated with UTI sagereviewcenter

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 ▪ Transitional (urothelial) epithelial cells

o Medium (20 – 30 um), polyhedral shaped cells with small central nuclei
(maybe bi-nucleated)
o Appear as having round or pear shaped contours and moderate
cytoplasm; may swell to spheroidal shape
o Originate from transitional epithelial lining of the renal pelvis, ureter,
urinary bladder and proximal urethra
o Few are seen in normal urine; elevated amounts may be associated with
UTI and large clumps suggest possible carcinoma

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 ▪ Renal Tubular epithelial cells

o Small (14-60 um), oblong or egg-shaped cells with large nuclei

may be centric or eccentric
o Appear to have coarsely granular eosinophilic cytoplasm
o Originate from proximal and distal convoluted tubules
o Presence may be associated with acute tubular necrosis, kidney
infection, or drug/heavy metal toxicity

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 ▪ Oval fat bodies

o Renal tubular cells or WBCs that have absorbed lipids

o Highly refractile and produce a characteristic maltese cross
appearance with polarized light
o Extremely significant finding; seen in lipid nephrosis and terminal
kidney disease

▪ Clue cells

o Squamous epithelial cells covered with coccobacilli; most

common pathogen is Gardnerella vaginalis
o Associated with bacterial vaginosis

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Red Blood Cells “Ghost cells”

▪ Small (6-8um), biconcave disc with central pallor

▪ May appear swollen in hypertonic urine and crenated in hypertonic urine
▪ Empty rbc membranes, referred to as ghost cells may be seen from lysed cells in
alkaline urine
▪ Elements that may be confused with red blood cells includes droplets, crystals
(urates) and yeast
o Correlate with occult blood test pad
o Addition of glacial acetic acid may assist in differentiation as RBC will lyse
▪ Reference Interval: 0 – 5 rbcs/hpf
▪ Increased presence maybe associated with:
o Renal disease such as glomerulonephritis, lupus nephritis, kidney stones,
tumors, and trauma
o Lower urinary tract disease such as acute and chronic infection, tumors
and strictures
o Extra renal disease such as acute appendicitis

White Blood Cells

▪ Small (10-12 um), spherical cells, appearance of a nuclei, cytoplasm, and

granules dependent on type
▪ May swell in alkaline or hypotonic urine; referred to as “glitter cells” due to
Brownian movement of granules in the cytoplasm
▪ Differentiate from renal tubular epithelial cells which have larger nuclei;
correlate with leukocyte esterase
▪ Reference Interval: 0 - 8 WBCs/hpf

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 ▪ Increased presence maybe associated with lower and upper UTIs or prostatitis

Urine Casts
▪ Cylindrical structures primarily composed of uromodulin (Tamm Horsfall
mucoprotein) that form in the lumen of renal tubules
▪ When present, albumins and globulins may contribute to cast formation by
combining with uromodulin
▪ Conditions that increase urine cast formation include:
o Dehydration or increased concentration of the urine
o Increased acidity of the urine
o High protein concentration
o Urinary stasis or obstruction

Hyaline Cast

o Formed in the lumen of the distal convoluted tubules or collecting

ducts; serves as a matrix of all casts
o Appears as pale, smooth cylinders with rounded ends and low
refractive index
o Narrow casts form in the convoluted tubules while broad casts
form in the collecting ducts
o Few hyaline casts may be present in healthy individuals
o Increased numbers may be associated with renal disease

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 Red Blood Cell Cast

o Hyaline cast with embedded rbcs in the matrix; rbcs must

be clearly identifiable sagereviewcenter
o Presence of clinically significant and associated with
glomerular disease or damage (e.g., acute
glomerulonephritis)
Hemoglobin Cast
o Rbc cast in which the red blood cells have ruptured;
appears reddish brown due to acid hematin formation
o Associated with glomerular disease or damage (e.g.,
acute glomerulonephritis)

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 White Blood Cell Cast

o Hyaline cast with WBCs embedded in the matrix

o Associated with renal inflammation or infection such as
acute pyelonephritis

Renal Tubular epithelial cells cast

o Hyaline cast with renal tubular epithelial cells embedded

in a matrix
o Form as a result of stasis and necrosis of the tubules

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 o Seen in severe chronic disease, exposure to nephrotoxic
agents or viruses and rejection in kidney transplants

Granular casts

o Thought to be the result of degeneration of cellular casts

components (eg. RBCs, WBCs)
o Progressive cellular deterioration leads to the appearance
of coarse granules which transition into fine granules
o Associated with prolonged renal disease

Waxy Cast

o Appears smooth, homogenous cylinders with blunt,

broken ends and cracked or serrated edges, highly
refractive
o Associated with severe chronic renal failure, malignant
hypertension, diabetic nephropathy, acute renal disease,
and renal transplant rejection

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 Fatty Cast

o Cast have incorporated either free fat droplets or oval fat

bodies
o If cholesterol is present, the droplet will demonstrate
“Maltese cross” appearance under polarized light
o droplets which consist of triglycerides or neutral fat will
not polarize light but will stain with Sudan III or Oil red O

Crystals seen in Acidic Urine

Calcium oxalate

▪ Dihydrate form
o Colorless octahedrons that are describe as “envelopes” and do
not polarize light
o Predominate in urine from patients with diets rich in oxalic acid;
most common cause of kidney stones
▪ Monohydrate form
o Described as dumbbells or rings
o Predominate in urine from patients who have ingested ethylene
glycol (anti-freeze)

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 Uric acid

▪ Colorless, rhombic plates that also appear as rosettes, wedges and

needles; appear as multicolored under polarized light
▪ Associated with gout or treatment with chemotherapy

Amorphous urates

▪
▪ Brown-yellow granules that resemble sand
▪ Presence in urine is considered clinically insignificant and
generally associated with old specimens

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Crystals seen in Alkaline Urine
Triple Phosphate

▪ Colorless, prisms that are described as” coffin lids” and

demonstrates birefringence under polarized light
▪ Presence in urine is considered clinically insignificant

Ammonium biurate

▪ Yellow-brow, spicule-covered spheres that are described

as “thorny-apples”
▪ Presence may be clinically significant if formed in vivo
(dehydration)

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 Amorphous phosphates

▪ Fine, colorless granules that resemble sand

▪ Presence is considered clinically insignificant and generally
associated with refrigeration
Cystine

▪ Colorless hexagonal plates that do not polarize light; maybe layered or

laminated
▪ Associated with inborn error of metabolism such as hereditary cystinosis
or cystinuria

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 Cholesterol

▪ Colorless, rectangular plate with notched corners that often appear

layered; demonstrate weak birefringence
▪ Associated with nephritic and nephrotic conditions or lymphatic damage
▪ Differentiate with radiographic contrast media based on specific gravity

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 Bilirubin

▪ Mostly commonly appears as small clusters of fine yellow-brown needles,

but may also form granules
▪ Associated with hepatic disorders

Tyrosine

▪ Colorless, fine needles that often appear in clusters or sheaves

▪ Associated with inborn errors of metabolism such as tyrosinemia and in
certain liver disorders in which amino acid metabolism is impaired

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 Leucine

▪ Yellow brown spheres with concentric circles that are birefringent under
polarized microscopy
▪ Associated with inborn errors of metabolism such as maple syrup disease

OTHER URINARY ELEMENTS

Yeast

▪ Colorless, ovoid cells that may show budding or development of

pseudohyphae; refractile when viewed with brightfield microscopy
▪ Maybe confused with red blood cells; correlate with occult blood test pad
result and look for budding
▪ Presence may indicate UTI. But is most likely a result of vaginal secretion
contamination

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 Bacteria

▪ Rod-shape bacilli are most frequently observed (E. coli) but cocci may
also be encountered (Staphylococcus spp)
▪ Bacteria maybe present due to UTI or contamination during collection;
correlate with nitrite pad test result.
Trichomonas

▪ Colorless, turnip-shaped flagellates with 4 anterior flagella, a posterior

axostyle, and an undulating membrane
▪ May be confused with WBCs or RTEs; compare with leukocyte esterase
and look for undulating membranes movement

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 Sperm

▪ Male reproductive cells with oval heads, and long thin tails
▪ Presence in urine is clinically insignificant unless patient is
considered part of vulnerable population
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CLINICAL SIGNIFICANCE

Diabetes Insipidus

▪ Due to lack of Antidiuretic hormone (ADH)

▪ Characterized by polyuria, polydipsia pale yellow urine, and a LOW
SPECIFIC GRAVITY

Diabetes Mellitus

▪ Due to lack of insulin or poor response to insulin

▪ Characterized by hyperglycemia, polyuria, polydipsia, pale-yellow urine,
glucosuria and HIGH SPECIFIC GRAVITY

Renal glycosuria
▪ Characterized by glucosuria despite a normal or decrease fasting blood
glucose concentration
▪ Due to poor renal tubular reabsorption of glucose

Galactosemia
▪ Due to inability to metabolize galactose
▪ Characterized by positive Clinitest

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 Jaundice
▪ Due to i9ncrease heme catabolism (hemolytic anemia, transfusion
reaction, acute hepatitis, hepatobiliary obstruction
▪ Characterized by increased concentrations of bilirubin (unconjugated or
conjugated) and/or urobilinogen; bilirubin crystals

UTI
▪ Most common pathogen is Escherichia coli
▪ Characterized by frequent painful urination (dysuria)
▪ Chemical examination may be positive for occult blood, nitrites, and
leukocyte esterase
▪ Microscopic examination may reveal presence of RBCs, WBCs, and
bacteria; epithelial cells may also be present.

AMNIOTIC FLUID

SYNTHESIS & COMPOSITION

▪ Produced in early gestation by amnion and placenta and is a dialysate of fetal and
maternal plasma

FUNCTION
▪ Cushions the fetus throughout the pregnancy
▪ Facilitates the transport of nutrients and waste products between the fetus and
maternal plasma

VOLUME

▪ In early pregnancy (12 weeks) – 20 – 50 ml, rising to 800 – 1200 ml by 37 weeks

and it is replenished every 2 – 3 hrs
▪ Oligohydramnios - <800 ml – low volume; associated with congenital
malformation and intrauterine infection
▪ Polyhydramnios – abnormally high volume >1200 ml; associated with congenital
malformations and decreased fetal swallowing

SPECIMEN COLLECTION & HANDLING

Amniocentesis – sample obtained transabdominally by passing a needle through

mothers abdomen, through the uterine wall, and into the amniotic sac.

Transport & Storage

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 - Protect from light during transport to prevent photo-oxidation of
bilirubin.
- Maintain specimens for cell culture, genetic studies, and microbial or
viral culture at body temp (37C) or RT (20 – 25C)
- Assay specimens for fetal lung maturity (FLM) testing immediately or
store refrigerated (2 – 5C)

PHYSICAL EXAMINATION
Color
▪ Colorless, pale yellow, or yellow
▪ Dark yellow or amber may indicate increased bilirubin concentration
▪ Green color suggests fetal distress and the passage of meconium in utero
▪ Meconium is a mucus-like substance that forms in the fetal intestinal tract from
swallowed amniotic fluid and intestinal secretions.
▪ Pink or red color indicates the presence of intact red blood cells or hemoglobin,
which maybe the result of contamination during collection. Specimens that may
contain blood should be centrifuged immediately to remove intact red blood cells
because oxyhemoglobin may interfere with biochemical tests such as bilirubin
▪ Dark red brown may indicate fetal death

CLARITY
▪ Naturally decreases as gestation progresses because of the accumulation of
cellular and particulate matter (fetal hair, cells, and vernix)

CHEMICAL EXAMINATION

❖ DIFFERENTIATION FROM MATERNAL URINE

• Biochemical tests
o Creatinine and Urea are non-protein nitrogen
compounds produced at fairly constant rates that are
excreted solely by the kidney. Bothe compounds have
higher concentrations in urine compared to plasma.
Amniotic fluid concentrations are similar to plasma until
late pregnancy when fetal kidneys filter creatinine and
urea for excretion into amniotic fluid
o Total protein is found in significant amounts in amniotic
fluid whereas maternal urine should have essentially
none unless maternal renal disease is present
o Glucose which is found in amniotic fluid should not be
present in maternal urine unless the mother has
diabetes or renal disease.

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 ❖ AMNIOTIC FLUID BILIRUBIN or A450

Absorption spectrophotometry

Queenan Chart for interpreting Absorbance OD450

Liley Chart for interpreting Absorbance OD450

• Bilirubin has an optimal absorbance at 450 nm

(spectrophotometrically)
• In healthy individuals, the spectral curve is essentially a straight
line with decreasing absorbances between 365 nm and 550 nm.

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 • Bilirubin concentration is directly related to the severity of
hemolysis.
• Interpretation is based on gestational age using either the
Queenan (<27 weeks) chart or Liley chart (>27 weeks).

❖ FETAL LUNG MATURITY

• Performed to evaluate if the fetus will be viable outside the
mother’s womb if premature delivery is expected or necessary
because of fetal distress
• Not performed before 32 weeks gestation because all tests will
indicate fetal immaturity
• Starting at 20-24 weeks gestation, lamellar bodies are secreted by
fetal type II pneumocytes.
o Lamellar bodies contain pulmonary surfactants, such as
lecithin and sphingomyelin which alter the surface tension
of the alveoli, preventing their collapse during expiration
and reducing the pressure needed to reopen the alveoli
during inspiration.
o Phospholipids such as phosphatidylglycerol (PG) enhance
the spread of pulmonary surfactant across the alveoli.
o Both phospholipids (lecithin and sphingomyelin) can be
measured using Thin Layer chromatography (TLC) and
reported as L/S ratio.
o An L/S ratio of ≥2.0 is associated with fetal lung maturity
• PG is undetectable until 35 weeks gestation
o PG can be measures using TLC or an agglutination slide test
using polyclonal anti-PG antibodies
o A positive result is associated with fetal lung maturity
• The Lamellar Body Count (LBC) can be measured using the platelet
channel of an automated hematology analyzer.
o In uncentrifuged specimens, an LBC >5.0 x 10^4/ul indicates
fetal lung maturity whereas a value <1.5 x 10^4/ul indicates
fetal lung immaturity

❖ Alpha- Fetoprotein (AFP)

• AFP is a glycoprotein secreted in fetal serum with concentrations
reaching a peak during the 13 th week of gestation and then
declining until the fetus is full term
• High concentrations are associated with open neural tube defects,
fetal abnormalities, and fetal distress.
❖ Acetylcholinesterase (AChE)
• Performed as a confirmation when a positive AFP value is obtained.

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 • Normal amniotic fluid does not contain AChE and a positive result
is generally associated with an open neural tube defect.
CLINICAL SIGNIFICANCE
▪ Hemolytic disease of the fetus
- Increased hemolysis of fetal rbcs leads to an elevated bilirubin
concentration in amniotic fluid which can be measured
spectrophotometrically with A450 test.
▪ Neural Tube Defects (NTDs)
- Birth defects affecting the brain, spine or spinal cord; the 2 most
common NTDs are spinal bifida and anencephaly.
o Spinal bifida – spinal column does not completely close
which leads to nerve damage and paralysis.
o Anencephaly – brain and skull do not fully develop, leading
to still birth or death shortly after birth.
- NTDs can be diagnosed through imaging studies and prenatal
testing of amniotic fluid for AFP and AChE.
- Maternal blood may also be screened in the second trimester for
AFP, hCG, and estriol in what is referred as a “triple screen”
▪ Respiratory Distress Syndrome
- Neonates with immature lungs that do not have sufficient amount
of pulmonary surfactants
- Neonate may show symptoms such as cyanosis, apnea, and rapid
or shallow breathing
- If premature delivery is necessary, fetal lung maturity testing is
performed to evaluate if the lungs are mature enough to survive
outside the uterus and to determine the fetus risk of developing
respiratory distress syndrome.

CEREBROSPINAL FLUID (CSF)

❖ Approximately 70% is produced by choroidal cells lining the choroid plexus into
the 4 ventricles of the brain.
❖ Remaining 30% is produced by ependymal cells lining the brain and spinal cord.
❖ CSF is found in the subarachnoid space between the pia mater and arachnoid
mater.
❖ Function: serves to protect and cushion the brain and spinal cord while facilitating
transport of nutrients and waste products through CNS.
❖ CSF is collected by passing a needle between the 3 rd and 4 th or 4th and 5th lumbar
vertebra and into subarachnoid space.
❖ CSF collected tubes should be labeled numerically in the order to which they are
filled.

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 o 1st tube – used for chemical and immunologic testing because any
cellular or bacterial contamination from the collection procedure
will not interfere with the test.
o 2nd tube – used for microbiological testing such as gram stain and
culture
o 3rd tube – for cellular studies (Hematology)
❖ Testing should be performed as soon as possible.
o If testing is delayed, the specimen should be stored as follows:
Chemical or immunologic testing – frozen (-15 to -30C)
Microbiological testing – RT (20 – 25C)
Cellular studies – keep refrigerated (2 – 5C)
PHYSICAL EXAMINATION
❖ Colorless
❖ The presence of a small amount of blood from a traumatic tap may give the first
tube pink/red coloration, but these should decrease in subsequent tubes, with the
final tube appearing colorless.
o Xanthochromia – pink, orange or yellow coloration consistent across all
tubes
o Pink coloration – presence of hemoglobin, which may be associated with a
subarachnoid or intracerebral hemorrhage
o Yellow coloration- presence of bilirubin associated with
hyperbilirubinemia
o Orange color – may contain both hemoglobin and bilirubin
❖ CSF is clear
o Hazy/cloudy or turbid may contain an increased presence of white blood
cells (WBCs), RBCs, microorganisms, proteins, or other constituents.
o Milky appearance – indicates high lipid content
o Radiographic contrast media gives an oily appearance
CHEMICAL EXAMINATION
❖ GLUCOSE
o Healthy individual concentrations are approximately 60% - 70% of the
plasma concentration
o Elevated concentrations are not significant – probably due to
contamination with peripheral blood or hyperglycemia
o Decreased concentrations – clinically significant associated hypoglycemia,
impaired glucose transport, increased CNS glycolysis, meningitis,
metastatic carcinoma.
❖ PROTEIN
o NV : 15 – 45 mg/dl (0.15 – 0.45 g/dl)
o Elevated concentrations: damage to the blood brain barrier; increased
intrathecal synthesis; decreased reabsorption
o Decreased: fluid loss from damage to the dura matter; increased
reabsorption because of increased intracranial pressure
❖ ALBUMIN

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 o To assess the integrity of blood-brain barrier, a paired serum specimen is
collected, and the CSF serum albumin index is determined as follows.

CSF/serum albumin index = CSF Albumin (mg/dL)/ serum albumin (g/L)

CSF serum index of >9 suggests damage to the blood brain barrier and
increased permeability.

❖ Immunoglobulin G (IgG)
o To distinguish the cause of an elevated IgG concentration, a paired serum
specimen is collected and both specimens are analyzed for IgG and
albumin
o CSF IgG index is calculated as:

CSF IgG index = serum albumin (g/dL) X CSF IgG (mg/dL)

CSF albumin (mg/dL) serum IgG (g/d)

Elevated CSF IgG indices indicates increased intrathecal synthesis as seen

in multiple sclerosis or inflammatory disorders

Decreased CSF IgG indices may be associated with damage to the blood-
brain barrier.

Cut off = 0.85

❖ Protein Electrophoresis
o Performed on concentrated CSF and serum specimen
o 4 distinct bands:
Prealbumin (transthyretin), albumin, transferrin (Beta 1) and T
transferrin (Beta 2)
T transferrin confirms the presence of CSF which may assist in the
diagnosis of CSF rhinorrhea.

❖ Myelin basic protein (MBP)

o Found in myelin sheath surrounding the axons of nerves
o In MS and other demyelinating disorders, the myelin sheath is degraded
releasing MBP which can be measured and used to follow the course of
disease progression.
❖ Lactate
o Present in CSF as a result of anaerobic metabolism in the CNS
o NV : 10 – 22 mg/dL (1.1 – 2.4 mmol/L)
o 25 – 30 mg/dL (2.7-3.3 mmol/L) – viral meningitis
o >35 mg/dL (>3.9 mmol/L) – bacterial, fungal, or tubercular meningitis

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CLINICAL SIGNIFICANCE

Pleocytosis – increased WBC

Neutrophilic pleocytosis – bacterial meningitis

Lymphocytic pleocytosis – occurs in later stages of viral, tubercular, and fungal infections;
lymphocytes may also predominate in syphilitic meningitis

Monocytic pleocytosis – uncommon, seen in mixed pleocytosis associated with

tubercular, fungal, or bacterial infections.

Eosinophil pleocytosis – associated with parasitic, fungal infections, allergic reactions

Plasma cells in abnormal in CSF and may be seen in acute viral or chronic inflammatory
conditions and these cells are often associated with Multiple Sclerosis.

Malignant cells maybe present as a result of primary tumor, metastases or leukemia.

Meningitis

- inflammation of the meninges

Multiple sclerosis

- Autoimmune disease in which the myelin sheaths surrounding nerve fibers is deteriorated
leading to nerve damage.

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 SEROUS FLUID

Pleural
Pericardial
Peritoneal

Effusions – excess amount of fluid within a cavity; an effusion in the peritoneal cavity is called
ascites.

Categories of Effusion
Transudates – result of non-inflammatory process leading to an increase in hydrostatic
pressure or decrease in oncotic pressure

- Clear, pale-yellow to yellow fluids with viscosity similar to that of

serum
Exudates – result of an inflammatory process causing an increase in endothelial
permeability or a decrease in lymphatic absorption.
- Cloudy, and may be yellow, green, pink, or red.

Clarity
Chylous – appear milky after centrifugation because of the presence of chyle and may
be the result of an obstruction or damage to the lymphatic system
Pseudochylous – similar to Chylous but are the result of a chronic effusion and cellular
breakdown.

Chemical Examination
Total Protein and Lactate Dehydrogenase
-to differentiate transudate from exudate
- exudates require further testing while transudates generally do not merit
further analysis

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Glucose
Simultaneous measurements of serum and serous glucose may be performed on
exudates.

Serous fluids with glucose concentrations <60 mg/dL (<3.3 mmol/L) or a difference
between the serous and serum glucose concentrations of >30 mg/dL (>1.6mmol/L)
identify an exudative process.

Conditions associated with low serous glucose concentrations include bacterial

infection, malignancy, and rheumatoid arthritis.

pH
Can aid in deciding treatment options for patients with parapneumonic effusions

PH >7.3 – prompts for treatment with antibiotics only

PH <7.3 – require placement of a drainage tube in addition to antibiotics.

Carcinoembryonic antigen (CEA)

Used in the evaluation of pleural and peritoneal fluids with possible carcinoembryonic
antigen – producing tumors.

Microscopic
>1000 WBC’s/ul - transudates
<1000 WBC’s/ul – exudates

SYNOVIAL FLUID

- formed by the secretion of synoviocytes (enzymes and hyaluronic acid ) and ultra-filtration of
the plasma across the synovial membrane

- serves as a lubricant and source of nutrients for the articular cartilage

Color

Colorless – pale yellow Normal

Pink or red discoloration Presence of blood because of traumatic
collection
Yellow Associated with non-inflammatory process
Yellow-white Suggestive of inflammatory process
Yellow-green Inflammatory process
Red or brown Hemorrhagic conditions

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Clarity
-normal synovial fluids are clear
- hazy, cloudy, or turbid fluids are associated with inflammation or infection
-The presence of rice bodies, composed of collagen and fibrinous tissue, are most commonly
seen in patients with rheumatoid arthritis.
- Ochronotic shards, which are pieces of pigmented cartilage, maybe seen in the synovial fluid
of patients with alkaptonuria

Viscosity
- assessed using string test in which a drop of fluid is expelled from the syringe and the
resultant strength is measured; normal fluid forms a string at least 4 cm in length before
breaking.

hyaluronidase is added for highly viscous specimen before proceeding with testing.

Chemical Examination
Mucin Clot Test
- performed by adding few drops of acetic acid to an aliquot of synovial fluid to
promote clot formation.
- Poor clot formation is associated with inflammatory conditions such as
rheumatoid arthritis.
Glucose
- simultaneous measurement of plasma and synovial glucose
- normally difference is <10 mg/dL (0.5mmol/L)
-inflammatory conditions yield a difference of 20 -40 mg/dL (1.1 – 2.2
mmol/L)
-non-inflammatory conditions yield difference of 20 – 40 mg/dL (0.5 – 1.1
mmol/L)
- Septic conditions demonstrate differences >40 mg/dl (>2.2 mmol/L)

Uric Acid
- plasma and synovial fluid uric acid concentrations are normally equivalent

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Microscopy
- birefringent appear as bright objects in dark background
- Using a compensating polarizing microscope with a red compensator allows
differentiation of positive and negative birefringence sagereviewcenter

Monosodium Urate (MSU) crystals

- Needle-like shape with strong negative birefringence.

- using a red compensator they appear yellow when their longitudinal axis is
parallel to the plate and blue when it is perpendicular to the plate.
-- MSU crystals are associated with increased purine metabolism as occurs in
gout.

Calcium pyrophosphate dihydrate (CPPD) crystals

- CPPD crystals have rodlike or rhombic shape and weak positive birefringence
- Using a red compensator, they appear blue when their longitudinal axis is
parallel to the plate and yellow when it is perpendicular to the plate.
-CPPD crystals associated with a group of disease known as pseudogout.

Clinical Significance
Gout
- results in increased purine metabolism leading to elevated blood and synovial
concentrations of uric acid
- causes pain and swelling in the joints, predominantly affecting the great toe.

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 - increased uric acid plus presence of MSU

Pseudogout
- causes pain and swelling in the joints, predominantly affecting the knee
- presence of CPPD crystals sagereviewcenter
Arthritis
- classified as inflammatory, non-inflammatory, septic or hemorrhagic
- color, clarity, wbc concentration, wbc distribution, plasma to fluid glucose
difference and gram stain aids in differentiation

SWEAT
Synthesis and Function
-produced by the eccrine glands
Why do we sweat?

Sweat Chloride Test

“Gold standard” for diagnosis of cystic fibrosis
- specimens are collected using pilocarpine nitrate iontophoresis
- specimen is then analyzed using chloridometry to determine chloride
concentration

Cystic Fibrosis
- autosomal recessive disease caused by mutations in the CFTR gene
- causes abnormal electrolyte and mucous secretion, leading to an elevated
sweat chloride and abnormally viscous secretions throughout the body.
- Mortality rate is most commonly the result of pneumonia

VAGINAL SECRETIONS
- collected to evaluate the risk of premature delivery in pregnancy

Fetal Fibronectin
- Glycoprotein found in the cells joining the placenta to the uterine wall
-between the 24 th to 35th weeks of pregnancy, fetal fibronectin should be undetectable
in vaginal secretions
- During labor, fFN is released and can be detected in cercovaginal secretions
- pregnant with elevated fFN concentration have a higher risk of premature delivery

Placental alpha-microglobulin -1 (PAMG-1)

- glycoprotein found in amniotic fluid
- presence in cercovaginal secretions is associated with premature rupture of
membranes and risk of premature delivery

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 -PAMG-1 levels may be measured using a lateral-flow, solid-phase
immunochromatographic assay.

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