MEDT 15 ANALYSIS OF URINE AND BODY FLUIDS 1

CHAPTER 1: INTRODUCTION TO URINALYSIS

 Urine Analysis – beginning of lab medicine; found in caveman drawings, Egyptian hieroglyphics, early physicians

HISTORY
500 BCE Hippocrates Wrote a book on uroscopy
Middle Ages — Intensive training of physicians on uroscopy
1140 — Use of color charts for significance of 20 different colors
1627 Thomas Bryant Published a book about charlatans called “Pisse Prophets” or “Medical Licensure Laws”
1694 Frederik Dekkers Discovery of albuminuria by boiling urine
Anton van Leeuwenhoek Invention of the microscope led to the examination of the urinary sediment and the development of
1700s
Thomas Addis the “Addis count”
1827 Richard Bright Introduced urinalysis as a part of a doctor’s routine patient exam
1864 Thudichum Named “urochrome”
1930s — Too many comlplex tests caused the impracticality of the urinalysis
Modern Day — Technology “resaved” routine urinalysis, remaining as an integral part of the patient exam

URINE COMPOSITION
 Urine is made of 95% water, 5% solutes w/variations due to dietary intake, physical activity, body metabolism,
endocrine function

Urea Major organic component, product of protein and amino acid metabolism
Creatinine Product of creatine metabolism by muscles
Uric acid Product of nucleic acid breakdown in food and cells
Other organic components Hormones, vitamins, medications, etc.
Chloride Major inorganic component, found in combination with sodium and many other inorganic substances
Sodium Major salt (combined with chloride)
Potassium Combined with chloride and other salts
Phosphate Combined with sodium as blood buffer
Ammonium By-product of protein metabolism, serves as a blood buffer
Calcium Combined with chloride, sulfate or phosphate
Magnesium Combined with chloride, sulfate or phosphate
Formed elements Cells, casts, crystals, mucus and bacteria (not part of the original plasma filtrate)

 What should a medical laboratory scientist do if there is a necessity to determine if a specimen is urine?
Test for the major component of urine (urea and creatine).

URINE VOLUME
 Factors Influencing Urine Volume  Normal Urine Output (Adults)
1. Fluid intake – Average: 1200-1500 Ml
2. Fluid loss from non-renal sources (e.g sweat) – Considered normal: 600-2000 mL
3. Variations in ADH secretion – Night: ~400 mL
4. Need to excrete solutes (e.g. glucose, salts)

Suppression of ADH:
Increase in urine output: >2500
1. Diabetes mellitus – SG = ?
Polyuria mL/day in adults;
2. Diabetes insipidus – SG = ?
> 2.5-3.0 mL/kg/day in children
3. Diuretics, caffeine, alcohol
Reduction in bladder capacity:
Increase in nocturnal urine 1. Pregnancy
Nocturia
output: approximately >500 mL 2. Bladder stones
3. Prostate enlargement
1. Dehydration: vomiting, diarrhea, perspiration, severe burns
Decrease in urine output: >400
Oliguria 2. Blockage: kidney stones or tumors
mL/day in adults
3. Low renal blood flow: shock, heart problems
Anuria Complete cessation of urine flow Same as with oliguria (more severe)

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CHAPTER 2: RENAL STRUCTURE AND FUNCTION

FUNCTIONS OF THE KIDNEYS Interlobular vein  Arcuate vein  Intelobar
1. Elimination of metabolic wastes vein  Renal vein (see Figure 2)
2. Homeostasis: acid-base balance, electrolyte c. Inferior vena cava
balance, organic compounds, blood pressure 4. Nephron
3. Production and regulation of hormones:  The functional unit of the kidney
a. Erythropoietin – produced by the peritubular  1-1.5 million nephrons comprise each kidney (2-3 M
fibrobalsts of the kidenys in response to low O2 per person)
levels  Consists of the renal corpuscle and a tubular
b. Renin – produced by the juxtaglomerular system 30-40 mm in length
apparatus of the kidneys; enzyme secreted by the a. Cortical nephrons (85%) – main function:
kidneys; part of the system that regulates blood removal of waste products and the reabsorption
pressure of nutrients
c. Prostaglandin – produced by the kidneys from b. Juxtamedullary nephrons (15%) – main
essential fatty acids for increase in renal blood function: concentration of urine
flow, sodium and water retention, as well as renin
release RENAL FUNCTION
d. Parathyroid Hormone (PTH) – promotes  Homeostatic functions and the excretion are
calcium reabsorption controlled in the renal blood flow by the following
e. Vitamin D – activated by the kidneys in response renal functions:
to PTH 1. Renal Blood Flow
f. Aldosterone – promotes sodium reabsorption 2. Glomerular Filtration
g. Antidiuretic Hormone (ADH) – promotes water 3. Tubular Reabsorption
reabsorption 4. Tubular Secretion

RENAL STRUCTURE (see Figure 1) Renal Blood Flow

1. Kidneys – two bean-shaped organs on either side of  Human kidneys receive 25% of the blood pumped
the aorta at the posterior, upper abdominal region through the heart which is equal to an average total
a. Cortex – outer layer consisting of the renal renal blood flow of 1,200 mL of blood per minute or a
corpuscles, as well as the PCT and DCT of nephrons total renal plasma flow of 600-700 mL of plasma per
b. Medulla – inner layer consisting of the Loops of minute through the renal artery.
Henle and the collecting ducts of nephrons  Each glomerulus is supplied with blood by an afferent
2. Structures for urine flow: arteriole from an interlobular artery.
a. Calyx – structure where the collecting ducts come  An efferent arteriole carries blood from the
together glomerulus to either the vasa recta or the peritubular
b. Renal pelvis – cavity-area that is an expansion of capillaries before returning it to the renal vein.
the ureter  The smaller diameter of the efferent arterioles (as
c. Ureter – muscular tube that connects the kidneys compared with the afferent arterioles), incorporate
to the bladder the glomerular capillary pressure which aids the
d. Urinary bladder – storage of urine glomerular filtration.
e. Urethra – excretes urine  The peritubular capillaries surround the PCT and the
3. Structures for blood flow: DCT, providing for the immediate reabsorption of
a. Abdominal aorta essential substances from the fluid in the PCT and
b. Renal artery  Segmental artery  Interlobar adjustment of the urinary composition in the DCT.
artery  Arcuate artery  Interlobular artery  The vasa recta, on the other hand, are capillaries that
 Afferent arteriole  Glomerulus  Efferent parallel the shape of the loops of Henle of the
arteriole  Peritubular capillaries/Vasa recta  juxtamedullary nephrons.

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 Major exchanges of salts and water take place in this arteriole and the macula densa in the distal
area, maintaining the osmotic gradient (salt convoluted tubule (DCT) (see Figure 4)
concentration) in the medulla, which is necessary for  Low plasma sodium content  Decreased water
renal concentration. retention  Decrease in BP  Detected by JGA
 JGC releases Renin  angiotensinogen in the
Glomerular Filtration blood turned to angiotensin I  Angiotensin
 The glomerulus consists of a coil of approximately Converting Enzyme (ACE) in the alveoli of the lungs
eight capillary lobes (capillary tuft). Its walls are activate it to angiotensin II
known as the glomerular filtration barrier.  Angiotensin II corrects renal blood flow through:
 It is located within the Bowman’s capsule which forms – Vasodilation of the afferent arterioles and
the beginning of the renal tubule/PCT. constriction of the efferent arterioles
 It served a non-selective filter of plasma substances – Stimulation of sodium and water reabsorption in
with molecular weights less than 70,000 the PCT
– Triggering the release of aldosterone from the
FACTORS AFFECTING GLOMERULAR FILTRATION adrenal cortex and ADH from the hypothalamus,
1. Cellular Structure of the Glomerulus (Figure 3) to stimulate the reabsorption of sodium and
 Plasma must pass through three glomerular water, respectively in the DCT and the collecting
filtration barriers: duct
a. “Fenestrated” capillary wall
membrane/endothelium: has increased  Because of the above mechanisms, a glomerular
permeability but do not allow the passage of filtration rate of 120 mL/minute through the 2-3 M
large molecules glomeruli of the kidneys happen
b. Basement membrane (basal lamina)  The glomerular filtrate is a cell and protein-free
c. Visceral epithelium epithelium of Bowman’s ultrafiltrate of plasma. It is iso-osmotic (275-300
capsule: thin membranes covering the filtration mOsm/L) with plasma, has a pH of 7.4 and has a
slits formed by the intertwining foot processes of specific gravity of 1.010 +/−0.002
the podocytes  Substances that is a part of the glomerular filtrate
 In addition to these structures that prohibits include the following:
filtration of large molecules, the barrier contains a  Water
“shield of negativity” that repels positively  Salts (sodium, chloride, potassium, calcium,
charged molecules that are small enough to pass bicarbonate, etc.)
through  Glucose
2. Glomerular Pressure/Hydrostatic Pressure =  Amino acids
75 mmHg (see Figure 3)  Urea
 Results from the smaller diameter of the efferent
arterioles, as well as the glomerular capillaries Tubular Reabsorption
 Necessary to overcome:  The process of the removal of substances from the
 Pressure from the fluid within the Bowman;s glomerular filtrate and returned to the blood in the
capsule peritubular capillaries
 Oncotic pressure of infiltered plasma proteins in  Proximal Convulated Tubule (PCT)
the glomerular capillaries – Reabsorbs most of the essential substances from
 Autoregulatory mechanism from the the glomerular filtrate:
Juxtaglomerular apparatus maintains the  All glucose (if levels do not reach the renal
glomerular pressure at a constant rate regardless threshold)
the of the systemic blood pressure fluctuations  Almost all amino acids, vitamins and proteins
3. Renin-Angiotensin-Aldosterone System (RAAS)  Water, sodium and chloride
 Regulates blood flow to and within the glomerulus  Variable amounts of urea , uric acid and ions
in response to changes in (a) blood pressure and (b) (Mg2+, Ca+, K+ and HCO3-)
plasma sodium content – Primary site of the excretion of waste substances
 Monitoring is done by the Juxtaglomerular that do not pass through the glomerulus:
apparatus: juxtaglomerular cells in the afferent  Renal tubular cell metabolism products (H )
+

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 Drugs like penicillin – Knowledge of plasma level and renal threshold
 Distal Convulated Tubule (DCT) – secretion of of a substance is useful to distinguish between
H+, K+ NH4 excess solute filtration and renal tubular damage
 Descending Loop of Henle (DLOH) – freely (e.g. Fanconi syndrome)
permeable to water (thus, reabsorbs water) but not to
other solutes  What does it mean if glucose appeared in the
 Ascending Loope of Henle (ALOH) – urine of a patient with normal blood glucose
level?
impermeable to water but reabsorbs sodium and
chloride Water PCT, DLOH, CD
 Collecting Duct (CD) – drains the urine from each Passive
Sodium ALOH
nephron, joining other collecting ducts, forming a Transport
Urea PCT, ALOH
papillary duct to carry urine to a calyx and then to the Salts, glucose, amino acids PCT
Active
renal pelvis Transport
Sodium PCT, DCT
 The latter portions of the DCTs and the collecting ducts Chloride ALOH
are responsible for the final adjustment of the pH,
osmolality and electrolyte content of urine, including Countercurrent Mechanism
the regulation of substances still presence in the  Selective reabsorption process occurring in the loop
filtrate of Henle (and vasa recta) to maintain the osmotic
– Reabsorption of sodium and water due to the action gradient of the medulla which is important for the
of aldosterone and ADH, respectively final concentration when it reaches CD
 Concentration starts as water is removed by
URINE REABSORPTION MECHANISMS AND CONCENTRATION osmosis in the DLOH, while Na and Cl are
1. Passive Transport reabsorbed in the ALOH. The water-impermeable
2. Active Transport walls of the ALOH prevents the excessive water
3. Countercurrent Mechanism (Tubular Concentration) reabsorption as the filtrate passes through the
4. Mechanism of ADH/Vasopressin (Collecting Duct highly concentrated renal medulla
Concentration  Actual concentration of the filtrate leaving the
ALOH is lower than that of plasma
Passive Transport  Reabsorption of sodium continues at the DCT under
 Movement of molecules across a membrane due to the control of aldosterone
variance in the concentration or electric potential in
the opposite sides of the membranes Mechanism of ADH/Vasopressin
 Just as the aldosterone production is controlled by
Active Transport the body’s sodium concentration, ADH production is
 Combination of reabsorbed substances with a controlled by body hydration
carrier protein in the renal tubular epithelial (RTE)  Homeostasis (especially of body chemicals) is the
cells final factor of urine volume and concentration:
 Substance is transferred across the cell membrane  Increased body hydration = Decreased ADH =
and back to the blood by the electrochemical Increased urine volume
energy created by the interaction  Decreased body hydration = Increased ADH =
 Maximal re-absorptive capacity (Tm) is reached if a Decreased urine volume
normally completely reabsorbed substance reached
an abnormally high level and the substance starts Tubular Secretion
appearing in urine.  Process of the passage of substances from the blood
 This plasma level at which active transport stops is in the peritubular capillaries to the tubular filtrate
termed as the Renal Threshold  Primary site is the PCT
– Glucose has a renal threshold of 160 to 180
mg/dL FUNCTIONS OF TUBULAR SECRETION
– Other threshold substances: amino acids, 1. Elimination of unfiltered unnecessary substances
sodium, potassium, chloride, ascorbic acid a. Waste products
b. Foreign substances

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 Most medications cannot be filtered by the 4. Availability to the body
glomerulus due bonding with plasma proteins 5. Availability of tests to analyze the substance
 When these protein-bound substances enter the
peritubular capillaries, they develop strong CLEARANCE TESTS
affinity to the tubular cells and dissociate from 1. Urea Clearance
their carrier protein; thus, causing the tubular 2. Inulin Clearance
cells to transport them into the filtrate 3. Creatinine Clearance
2. Regulation of acid-base balance 4. Estimated Glomerular Filtration Rate (eGFR)
 To maintain the normal blood pH of 7.4, the blood 5. Cystatin C
must buffer and eliminate the excess acid formed 6. Beta-2-Microglobulin
by dietary intake and body metabolism 7. Radionucleotides
 Bicarbonate (see Figure 5)
 Phosphate (see Figure 6) Urea Clearance
 Ammonia (see Figure 7)  Earliest test due to presence in all urine specimen
 Should there be a need for additional elimination of and existence of routine chemical analysis
hydrogen ions, the DCT and CD are both able to  ~40% of filtered urea is reabsorbed, thus NV were
produce ammonium ion adjusted to reflect the reabsorption
 All three mechanisms occur simultaneously at rates  Patients were hydrated to produce a urine flow of 2
determined by the acid-base balance in the bpdy. mL/min to ensure that reabsorbed urea will not
Disruption of secretory functions can result to reach more than 40%
metabolic acidosis or renal tubular acidosis
 Renal Tubular Acidosis – inability to produce Inulin Clearance
acid urine in the presence of metabolic acidosis  A polymer of fructose, it is an extremely stable
substance that is not reabsorbed nor secreted by
RENAL FUNCTION TESTS the tubules
1. Glomerular Filtration/Clearance Test  Not a normal body constituent, thus it must be
2. Tubular Reabsorption/Concentration Test infused by IV at a constant rate throughout testing
3. Tubular Secretion and Renal Blood Flow Tests  The original reference method for clearance tests

Glomerular Filtration/Clearance Tests Creatinine Clearance

 Standard tests used to measure the filtering capacity  Waste product of muscle metabolism normally
of the glomeruli found at a relatively constant level in the blood
 Measures the rate at which the kidneys can remove  Neither reabsorbed nor secreted in the tubules
(clear) a filterable substance from the blood  Disadvantages:
 Clearance – volume of plasma from which a  Some creatinine is secreted by the tubules and
measured amount of substance can be completely secretion increases as blood levels rise
eliminated into the urine per unit of time expressed in  Chromogens present in plasma react in chemical
mL/min analysis. However, presence may help
counteract the falsely elevated rates
EXOGENOUS vs ENDOGENOUS PROCEDURES  Medication which inhibit tubular secretion of
EXOGENOUS ENDOGENOUS creatinine cause falsely low serum creatinine
 Test that requires an infused  Test substance that is  Bacteria will break down urine creatinine if
substance (i.e. inulin, already present in the body specimens are kept at room temperature for
radionucleotides) (i.e. urea, creatinine, B-2-
 Seldom the method of choice microglobulin, cystatin C) extended periods
 Heavy meat diet within the 24-hour period of

CRITERIA IN SELECTING CLEARANCE TEST SUBSTANCES urine specimen collection will influence the result
1. Neither secreted nor reabsorbed by the tubules
if the plasma specimen is drawn before the urine
2. Stability in urine during a possible 24-hour collection
collection period
 Accuracy depends on the accurate completeness
period
3. Plasma level consistency
of a 24-hour collection (greatest cause of error in

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clearance procedures are caused by improperly Cystatin C
timed urine specimens)  Small protein (MW 13,359) produced at a constant
 Not a reliable indicator in muscle-wasting rate by all nucleated cells
disease patients, persons involved in heavy  Readily filtered by the glomerulus, reabsorbed and
exercise, or athletes using creatinine broken down by the renal tubular cells
supplements  Not secreted by the tubules, and the serum
 Must be corrected for body surface area concentration can be directly related to the GFR
 Formula: 𝐶 = ×  Immunoassay procedures are available
𝑈𝑉 1.73
𝑃 𝐴
(see Figure 8)
 Normal Values  Good procedure for both screening and monitoring
 Creatinine Clearance (Male): 107-139 mL/min GFR
 Creatinine Clearance (Female): 87-107 mL/min  Recommend for pediatrics, diabetics, elderly, and
 Plasma Creatinine: 0.5-1.5 mg/dL critically ill patients
 Values are lower in older people  Advantage: independent of muscle mass
 Interpretation of GFR is dependent on two factors:  Measurement of both serum/plasma cystatin C and
1. Number of functioning nephrons creatinine can provide a more accurate information
2. Functioning capacity of the nephrons on a patient’s GFR

 How can you explain persons who live normal Beta-2-Microglobulin

lives with only one kidney?  Small protein (MW 11,800) that dissociate from
human leukocyte antigens at a constant rate
 Uses of Creatinine Clearance:  Rapidly removed from the plasma by glomerular
1. Determine the extent of nephron damage in filtration
known cases of renal disease  Sensitive enzyme immunoassays are available
2. Monitor the effectiveness of treatment designed  A rise in the plasma level has been shown to be a
to prevent further nephron damage more sensitive indicator of a decrease in GFR than
3. Determine the feasibility of administering creatinine clearance
medications that can build up to dangerous blood  Not reliable in patients with history of immunologic
levels if the GFR is marked reduced disorders or malignancy
4. Its value does not lie in the detection of early
renal disease Radionucleotides
 Exogenous and more labor intensive and costly
Estimated Glomerular Filtration Rate (eGFR) 125
 Injecting radionucleotides such as I-iothalamate
 Newer methods that do not require the collection of provides a method for determining GFR through the
timed urine specimens plasma disappearance of the radioactive material
 Uses of eGFR: and enables visualization of the filtration in one or
 In the past years, variety of formulas have been both kidneys
used and continue to be revised  Valuable to measure the viability of a transplanted
 Most commonly used = MDRD-IDMS-traceable kidney
formula:
𝐺𝐹𝑅 = 175 × 𝑠𝑒𝑟𝑢𝑚 𝑐𝑟𝑒𝑎𝑡𝑖𝑛𝑖𝑛𝑒 −1.154
× 𝑎𝑔𝑒 −0.203
Tubular Reabsorption/Concentration Tests
 Considerations:  Determine the ability of the tubules to reabsorb the
 Adjustments: x0.742 (female patient); x1.202 essential salt and water that have been non-
(black patient) selectively filtered by the glomerulus
 Designed to essentially equal the results that  Useful indicator of early renal disease as the loss of
compare to the reference body size (1.73 m2); tubular absorption capability is one of the first
thus, inaccurate for pediatric patients function affected in renal disease
 Most accurate when results are lower than 60
 As previously mentioned, the glomerular filtrate SG =
mL/min***
1.010; therefore, it is expected that final urine product
should be more concentrated. However, in the
performance of routine urinalysis, many specimens

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may be lower than 1.010 without any renal disease  Urine: Serum osmolality = 1.1
present, why?  Urine: Serum osmolality, controlled fluid ntake
and injection of ADH = 3:1 (see Figure 10)
(see Figure 9)  Uses of Osmolality
 Initial evaluation of renal concentrating ability
CONCENTRATION TESTS  Monitoring of the course of renal disease
1. Fishberg
 Monitoring fluid and electrolyte therapy
2. Mosenthal
 Differential diagnosis of hypernatremia and
3. Osmolality
hyponatremia
 Evaluation of the secretion of and renal response
Fishberg to ADH
 Patients were deprived of fluids for 24 hours
before measuring specific gravity FREE WATER CLEARANCE
 Measures the renal clearance of substance-free water
Mosenthal  Determine the ability of the kidney to response to the
 Comparison of the volume and specific gravity of
state of body hydration
day and night urine samples to evaluate  Osmolar clearance indicates how much water must be
concentrating ability cleared each minute to produce a urine with the same
osmolality as the plasma
 Both Fishberg and Mosenthal test are now obsolete  Comparison of the osmolar clearance with the actual
as the information provided by specific gravity urine volume excreted per minute, it can be
measurements are only useful as a screening determined whether the water being excreted is more
procedure. Quantitative measurement of renal or less the amount needed to maintain an osmolarity
concentrating ability is best assessed through the same as that of the ultrafiltrate
osmometry.  Formula: 𝐶𝑤𝑎𝑡𝑒𝑟 = 𝑣 − 𝐶𝑂𝑠𝑚 𝐶𝑂𝑠𝑚 =
𝑈
𝑂𝑠𝑚 ×𝑉
𝑃
𝑂𝑠𝑚

 Interpretation:
 Specific gravity is influenced by both the number
 0 – no renal concentration or dilution would be
and density (MW) of the particles while osmolality
taking place
measures only the number of particles in the
 -20 – less than the necessary water is being
solution
excreted (dehydration)
 Evaluation of renal concentration is concerned with
 +2.0 – excess water than necessary is being
small particles, primarily sodium and chloride
excreted
 On the other hand, molecules with large molecular
weight like urea do not contribute to the evaluation
Tubular Secretion and Renal Blood Flow Tests
of renal concentration; nonetheless, they
 Tests to determine both functions are closely related
contribute more to specific gravity
as total renal blood flow through the nephron must be
 Therefore, osmolality is performed for a more
measured by a substance that is secreted (from the
accurate evaluation of renal concentration
peritubular capillaries) rather than filtered (through
the glomerulus)
Osmolality
 Determined by measuring a property
PHENOLSULFONPHTHALEIN/PSP
mathematically related to the number of particles
 Interference by medications
in the solution (colligative property), then
 Elevated waste products in patients’ serum
comparing the yielded value with the value
 Necessity to obtain several very accurately timed
obtained from a known solution
urine specimens
 Instruments used:
 Possibility of producing anaphylactic shock
 Freezing point osmometer
 Vapor pressure osmometer
INDIGO CARMINE
 Normal Values:
 Used as confirmatory for unilateral kidney disease
 Serum osmolality = 275-300 mOsm
 Urine osmolality = 50-1400 mOsm

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PARA-AMINOHIPPURIC ACID (PAH)  Alkaline tides upon rising and postprandially
 Nontoxic substance that is loosely bound to plasma (around 2-8 PM)
proteins, permitting complete removal (except for a  Lowest pH is during the night
small amount that does not come in contact with  Measurement of mentioned parameters aids the
functional renal tissue) determination of defective “acid secretion”
 Infusion of PAH by IV should be monitored carefully;  Patients are primed with an acid load consisting of
thus, test is usually performed by specialized renal oral ammonium chloride
laboratories  Tests can be run simultaneously on either fresh or
 Formula: 𝐶𝑃𝐴𝐻 (𝑚𝐿/𝑚𝑖𝑛) =
𝑈(𝑚𝑔/𝑑𝑙 𝑃𝐴𝐻)× 𝑉(𝑚𝐿/ min 𝑢𝑟𝑖𝑛𝑒)
𝑃(𝑚𝑔/𝑑𝐿 𝑃𝐴𝐻)
toluene-preserved urine collected at 2-hour
 Normal Values: 600-700 mL/min (total renal plasma intervals
+
flow) or 1200 mL/min (total blood flow)  Free H (titratable acidity) is measured, followed
by the total acidity of the specimen
URINE PH, TITRATABLE ACIDITY AND URINARY AMMONIA  The ammonium concentration can be calculated as
 Normal person excretes approximately 70 mEq/day of the difference between the titratable acidity and
acid in the form H+, H2PO4-, NH4+ total acidity
 Diurnal variation of urine pH:

CHAPTER 3: SPECIMEN COLLECTION AND HANDLING

SPECIMEN HANDLING  Attached to the container, not the lid
 Urine is a biohazardous substance that require the  Should not become detached if the container is
observance of standard Precautions refrigerated or frozen
 Gloves should be worn at all times when in contact
with the specimen  A requisition form matching the information on the
label should always accompany specimens delivered
Containers
 Clean, dry, leak-proof Specimen Rejection
 Disposables should be used to eliminate the chance of  Unlabeled containers
contamination due to the improper washing  Nonmatching label and requisition form
 It is available in variety of shapes and sizes including  Contamination of specimens with feces, tissue, etc.
(but not limited to): (ask patient for another sample)
 Bag with adhesive for pediatric specimens  Containers with contaminated exteriors (ask patient
 Large containers for 24-hour specimens with a for another sample)
capacity of 2000 ml or 2 L mainly used for  QNS – quantity not sufficient (ask patient for another
creatinine clearance sample)
 Screw-top lids are less likely to leak than snap-on lids  Improper transportation/preservation (ask patient for
 Wide mouth and a wide, flat bottom another sample)
 Made of a clear material
 Recommended capacity of 50 ml  Never discard a specimen without notifying the
 Individually packaged sterile containers: supervisor and the personnel in-charge of the
 Microbiological urine studies collection.
 Suggested if more than hours elapse between
collection and analysis SPECIMEN HANDLING
 Specimens should be delivered to and tested in the
Labels laboratory within 2 hours from collection
Specimens should have proper label including:  If it is impossible to deliver specimen within 2 hours,
 Patient’s name it should be refrigerated or added with appropriate
 ID number chemical preservative
 Date and time of collection
 Additional info such as patient’s age, location, etc.

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Ideal Preservative  In choosing preservatives for specimens to be
 Bactericidal transported, always check with the receiving
 Inhibit urease laboratory.
 Preserve formed elements in the sediment  Refrigerated specimens must be returned to room
 Not interfere with chemical tests temperature before chemical testing

 Although an ideal preservative does not exist, a

proper preservative that best suits the needs of the
required analysis should be chosen.

CHANGES IN UNPRESERVED URINE

CHANGE ANALYTE CAUSE
Bacteria Multiplication
Nitrate Nitrate-reducing bacteria multiplication
Loss of carbon dioxide
pH
Increase Breakdown of urea to ammonia by urease-producing bacteria
Odor Breakdown of urea to ammonia by urease-producing bacteria
Color Oxidation or reduction of metabolites
Crystals Precipitation due to standing and cooling
Clarity Bacterial growth and possible precipitation of amorphous crystals
Glucose Cellular and bacterial utilization (glycolysis)
Ketones Bacterial metabolism and volatilization
Decrease Bilirubin Exposure to light/photo-oxidation to biliverdin
Urobilinogen Oxidation to urobilin
Intact RBCs, WBCs, and casts Disintegration due to dilute, alkaline urine
Trichomonas Immobilization or death

Story of Bacteria together they call themselves “the cast,” bilirubin who hates light,
There once this bacteria that lives in unpreserved urine. He best friend, urobilinogen, trichomonas, the only female in the
loves looking at the crystals on his yard. They grow and grow and group. But now, they’re gone! Bilirubin hates the light so much that
add color to his home (high crystals turbidity odor = low clarity). He when he saw it, he died. Urobilinogen was laughing hard at one of
has a goal and that is to buy a PHONE (pH, odor, nitrate). In order bacteria’s jokes, and then he inhaled a lot of oxygen then died. WBC
to buy a PHONE, he needs to work. His work is to breakdown urea and RBC who call themselves “the cast” drank too much dilute
to ammonia. He reduces nitrate to nitrite. Whenever he finishes his alkaline liquid and died. Trichomonas could have waited for
work, he will have a PHONE. The more he worked, the more PHONE bacteria. He could have saved her but she was with another man
he gets. He has lots of friends. WBC and RBC, when they’re (WBC). So bacteria let her die with him. Now bacteria is all alone.

URINE PRESERVATIVE
PRESERVATIVES ADVANTAGES DISADVANTAGES
 Does not interfere chemical tests
Refrigeration  Precipitates amorphous urates or phosphates
 Prevents bacterial growth for 24 hours
 Prevents bacterial growth and metabolism  Interferes with drug and hormone analysis
Boric acid
 Can be used for urine culture transport  Keeps pH at 6.0
 Excellent sediment preservative  Interferes with test for glucose, blood, leucocyte
Formalin
 Formerly used as Addis count preservative esterase, copper reduction
 Inhibits reagent strip tests for glucose, blood and
Sodium fluoride  Good preservative for drug analyses
leucocyte esterase
Commercial  Convenient when refrigeration is not possible  Know tablet composition to determine possible effects
preservative tablets  Controlled concentration to minimize interference on tests
 Contains collection cup, transfer straw, and culture and
Urine collection kits
sensitivity preservative tube or UA tube
 Cannot be used if urine is below minimum fill line
(insufficient urine)
 Sample stable at RT for 48 hours
C&S tube (gray top)  Note: Preservative is boric acid, sodium borate and
 Prevents bacterial growth and metabolism
sodium formate (thus disadvantages of boric acid
applies)
Urinalysis plus tube  Used on automated instruments  Refrigerate within 2 hours with no preservatives

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(yellow top)  Uses round or conical tubes
 Stable for 72 hours at room temperature
Preservative plus  Compatible with automated instruments  Cannot be used if urine is below minimum fill line
tube (cherry  Note: Preservative is sodium propionate, ethyl  Bilirubin and urobilinogen may be decreased if
red/yellow top) paraben, and chlorhexidine: uses either round or specimen is exposed to light and left at RT
conical tubes

TYPES OF SPECIMEN Catheterized Specimen

 Collected under sterile conditions by passing a
Random Specimen catheter through the urethra into the bladder
 Single/occasional specimen  Most commonly used for bacterial culture
 Collected anytime without any patient preparation  Can also be used to measure function of each kidneys
 Most commonly received specimen because of its ease  Not recommended anymore due to its painful
of collection and convenience to the patient procedure
 Used for routine screening to detect obvious  Used for bedridden patients or if patient is having
abnormalities difficulty voiding or urinating
 May show erroneous results from dietary intake or
physical activity prior to collection Midstream Clean-Catch Specimen
 Prior to collection, patient/collector is instructed for
First Morning aseptic collection technique (external genitalia is
 8 hour specimen thoroughly cleansed with a mild antiseptic solution;
 Collected immediately upon walking/rising and women: spread labia apart while voiding; male:
delivered to the laboratory within 2 hours withdraw the foreskin if necessary)
 Ideal routine screening specimen  Middle part of a single continued urination is collected
 Essential in preventing false negative pregnancy tests  Safer, less traumatic alternative to catheterized
and evaluation of orthostatic proteinuria specimen
 Concentrated specimen, assuring the detection of  Less contaminated by epithelial cells and bacteria
chemicals and formed elements that may not be  Used for both routine urinalysis and bacterial culture
present in a dilute random specimen
 If both routine UA and bacterial culture are requested
Timed Specimen on a specimen, which should be performed first?
 24-hour Specimen
 Collected within a 24-hour period
Suprapubic Aspiration
 Collection of urine by external introduction of a
 Patient begins and ends with empty bladder
 Specimen is refrigerated or kept on ice during the
needle (direct puncture) through the suprapubic region
24-hour collection period (abdomen into the bladder)
 It avoids vaginal and urethral contamination and can
 Used to measure solutes that changes
concentrations with diurnal variations and daily also be useful in getting urine from infants and small
activities children
 Provides a sample for bacterial culture (anaerobic)that
 Glucose Tolerance Specimen
 Collected with blood samples during a glucose
is completely free of extraneous contamination
 May also be used for cytologic examination
tolerance test
 Tested for glucose or ketone to aid in the
interpretation of a patient’s ability to metabolize a Prostatitis Specimen
 As with midstream clean-catch collection, the external
measured amount of glucose
 Afternoon Specimen
genitalia should be thoroughly cleaned
 Used to determine prostatic infection
 Collected between 2-4 pm for urobilinogen
1. Three-Glass Collection
determination
 Uses specimens:
 2-hour Post-prandial Specimen
 First urine passed
 4-hour Specimen
 Midstream portion

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 Prostate massage so that prostatic fluid will  More than 10-20 WBCs/HPF is considered
be passed in the remaining urine abnormal
 Uses three glass/bottles: (1) first urine, (2) mid-
stream portion, (3) urine after the massage of Pediatric Specimen
prostate  Soft, clear plastic bags with hypoallergenic skin
 Quantitative cultures are performed on all adhesive to attach to the genital area (routine)
specimen  Catheterization/suprapubic aspiration/clean-catch
 First and third specimens are examined procedure and sterile collection bag (culture)
microscopically
 In prostatic infection, the third specimen will  How often should you check if the applied bags have
have WBC/HPF count and bacterial count 10 times collected an ample amount of specimen?
that of the first specimen
 Second specimen serves as control for bladder
Drug Testing Specimen
 _____ part of the drug-testing program
and kidney infection
 Chain of Custody (COC) – process that provides the
 If the second specimen is positive, third
specimen result is considered invalid documentation of proper sample identification from
2. Pre- and Post-Massage Test time of collection to the receipt of laboratory results:
 Uses two bottles: (1) clean catch mid-stream
a standardized form
 Urine specimens must be tampered via dilution
urine. (2) urine after the massage of prostate
 Two specimens are collected equivalent to the
(addition of water), substitution (substituting with a
second (pre-massage) and third specimen (post- drug-free specimen at the time of collection), or
massage) of the three-glass alteration (ingesting/adding chemicals that cause
 Positive result significant bacteriuria in the post-
drugs in the specimen to be undetectable).
 Specimen requirements:
massage of greater than 10 times the pre-
 Witnessed or unwitnessed collection
massage count
 Immediately handled to the collector
3. Stamey-Mears test
 30-45 mL of urine in a container with 60 mL
 Consists of bacterial cultures of the initially
voided urine (VB1), mid-stream urine (VB2), capacity
 Urine temperature within 4 minutes: 32.5-37.7°C
express prostatic secretions (EPS), post-prostatic
 Urine color is inspected to identify contaminants
massage urine (VB3)
 Specimen should be labeled, packaged and
 Four glasses are collected VB1, VB2, EPS, VB3
 EPS is cultured and examined for WBCs
transported following laboratory protocols

CHAPTER 4: PHYSICAL EXAMINATION OF URINE

COLOR  Increased amounts produced in thyroid conditions
 Indication of the degree of hydration and urine SG and fasting states
 Specimen in a clear container is visually examined,  Increases in urine standing at room temperature
holding white background w/ adequate room lighting 2. Uroerythrin
 The lighter the color of the urine, the lesser is its  Pink pigment that is most evident in specimens that
content, and the lesser is its specific gravity have been refrigerated, resulting in the
precipitation of amorphous urates (where
Pigments Producing the Normal Colors of Urine uroerythrin attaches) and uric acid crystals
1. Urochrome 3. Urobilin
 Product of endogenous metabolism produced by the  Oxidation product of urobilinogen, a normal urinary
body at a constant rate under normal conditions constituent
(clear-yellow urine)  Imparts an orange-brown color to urine (not fresh)

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LABORATORY CORRELATION OF URINE COLOR
COLOR CAUSE CLINICAL/LABORATORY CORRELATIONS
COLORLESS Recent fluid consumption Commonly observed with random specimens
Polyuria-Diabetes insipidus Increased 24-hour volume and low specific gravity
PALE YELLOW Polyuria-Diabetes mellitus Elevated specific gravity glucose test result
Dilute random specimen Recent fluid consumption
Concentrated specimen Normal after strenuous exercise or in first morning specimen
DARK B-complex vitamins
YELLOW / Dehydration Fever or burns
AMBER / Bilirubin Yellow foam when shaken and positive chemical test results for bilirubin
ORANGE Acriflavine Negative bile test results and possible green fluorescence
Nitrofurantoin Antibiotic administered for urinary tract infections
Photo-oxidation of large amount of urobilinogen to urobilin
Urobilin
No yellow foam upon shaking unlike bilirubin
ORANGE-
Phenazopyridine (Pyridium) Drug commonly administered for urinary tract infections
YELLOW
Azo-gantrisin compounds May be mistaken as bilirubin due to yellow foam produced when shaken
Phenindione Anticoagulant, orange in alkaline urine, colorless in acid urine
YELLOW-
Bilirubin oxidized to biliverdin Colored foam in acidic urine and false-negative chemical test results for bilirubin
GREEN
GREEN Pseudomonas infection Positive urine culture
Amitriptyline Antidepressant
Methocarbamol (Robaxin) Muscle relaxant, may be green-brown
Clorets None
BLUE-GREEN
Indican Bacterial infections, intestinal disorders
Methylene blue Fistulas
Phenol When oxidized
Klebsiella / Providence
PURPLE
infection
RBCs Cloudy urine with positive chemical test results for blood and RBCs visible microscopically
Clear urine with positive chemical test results for blood; intravascular hemolysis = red
Hemoglobin
plasma
Clear urine with positive chemical test results for blood; muscle damage; more rapidly
Myoglobin
cleared from plasma and thus does not affect the color of plasma
PINK / RED
Beets Alkaline urine of genetically susceptible persons
Blackberries Red color in acidic urine
Menstrual contamination Cloudy specimen with RBCs, mucus, and clots
Rifampin Tuberculosis medication
Other medication Phenolphthalein, phenolsulfonphthalein, phenindione, phenothiazines
PORT WINE Porphyrins Negative test for blood, may require additional testing
Hemoglobin oxidation seen in acidic urine after standing, positive chemical test result for
Methemoglobin blood
RED-BROWN
Glomerular bleeding resulting to the conversion of hemoglobin to methemoglobin
Myoglobin
Homogentisic acid (alkaptonuria) Seen in alkalinized urine due to standing; specific tests are available
Melanini from melanogen
Urine darkens upon oxidation and reacts with nitroprusside and ferric chloride
(malignant melanoma)
BROWN /
BLACK Phenol derivatives Interfere with copper reduction test
Argyrols (antiseptic) Color disappears with ferric chloride
Methyldope or levodopa Antihypertensive
Metronidazole (flagyl) Darkens on standing, intestinal and vaginal infections

CLARITY TERM DESCRIPTION

 The transparency or turbidity of a urine specimen CLEAR No visible particulates, transparent
 Specimen in clear bottle is visually examined holding
HAZY Few particulates, print easily seen through urine
CLOUDY Many particulates, print burred through urine
against newspaper w/ adequate room lighting TURBID Print cannot be seen through urine
MILKY May precipitate or be clotted

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NON-PATHOLOGIC CAUSES OF TURBIDITY

CAUSES CLINICAL/LABORATORY CORRELATIONS
Squamous epithelial cells
More commonly seen in women (pregnant, after menstruation, unhygienic)
Mucus
Amorphous urates Pink (due to uroeythrin) or white precipitate in acidic urine
Amorphous phosphates
White precipitates in alkaline urine
Carbonates
Oxalates Precipitates seen in acidic, alkaline or neutral urine
Semen/Spermatozoa Not dissolved by dilute acetic acid, commonly observed in males, sometimes female
Fecal contamination
Radiographic contrast media Seen in acidic urine
Talcum powder
Vaginal cream

PATHOLOGIC CAUSES OF TURBIDITY

CAUSE CLINICAL/LABORATORY CORRELATIONS
RBCs Most commonly encountered
WBCs Correlate microscopically
Bacteria Seen in patients with bacterial UTI (bladder, kidney infection)
Yeast Less frequently seen in normal UA (large amounts indicate an infection,
Non-squamous epithelial cells kidney disease, or other serious medical conditions)
Abnormal crystals Correlate microscopically
Lymph fluid (chyluria) Causes milky appearance
Most commonly associated with parasitic infection (Wuchereria bancrofti)
Lipids (lipuria)
Can also be caused by non-infectious causes (lymphatic flow disorder)

SPECIFIC GRAVITY URINOMETER

 Indicator of the concentration of dissolved materials  Consists of a weighted float attached to a scale that
in urine, however, it is dependent not only upon the has been calibrated (20°C) in terms of urine specific
number of particles, but also upon the weight of the gravity
particles in the solution  Displaces a volume equal to its weight and has been
 The density of a solution compared with the density of designed to sink to a level of 1.000 in distilled water
a similar volume of distilled water at a similar at 20°C
temperature  Less accurate than other methods and not
 A measure of the density of the dissolved chemicals in recommended by the CLSI (Clinical & Laboratory
the specimen Standard)
 Influenced by both the number and size of the  Setbacks:
particles, thus large molecules contribute more to the  Large volume of urine is necessary
reading compared to small particles  Time-consuming
 Used to evaluate the kidney’s ability to concentrate  Needs correction for the following:
urine – Temperature – most urinometer are calibrated
 Useful in differentiating diabetes insipidus and to read at 20°C
diabetes mellitus  For every 3°C above the calibration
temperature, subtract 0.001 from the reading
Instruments Used to Measure SG  For every 3°C below the calibration
1. Urinometer temperature, add 0.001 from the reading
2. Harmonic Oscillation Densitometry – Protein – for each gram of protein present in the
3. Refractometer (indirect method) sample, subtract 0.003 from the reading
4. Falling Drop Method (direct methods) – Glucose – for each gram of glucose present in
5. Osmolality the sample, subtract 0.004 from the reading
6. Regent Strip

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HARMONIC OSCILLATION DENSITOMETRY  The falling time is electronically measured and
 Based on the principle that the frequency of a sound expressed as specific gravity
wave entering a solution changes in proportion to the
density of the solution OSMOLALITY
 Urine enters a glass tube with an electromagnetic coil  Specialized procedure not usually performed in the
at one end. After application of electric current sonic urine urinalysis department because it requires more
oscillation is produced which is proportional to the SG time and it is more expensive
of the urine. Temperature is corrected by a  Measured by comparing two solutions (unknown to
microprocessor known)
 Originally used in early automated urinalysis  Either measured by freezing point or by vapor
instruments but replaced by reagent strip analysis pressure depression
 Better indicator of the concentrating and diluting
REFRACTOMETER abilities of the kidneys because it is not affected by
 Measures the refractive index of urine (ratio of the density of solutes (glucose, protein, dextran,
velocity of light in air with the velocity of light in radiographic types)
solution)  Uses of osmolality:
 Uses a prism to direct a specific (monochromatic)  Initial evaluation of renal concentrating ability
wavelength of light against manufacturer-calibrated  Monitoring of the course of renal disease
specific gravity scale.  Monitoring fluid and electrolyte therapy
 The concentration of the specimen determines the  Differential diagnosis of hypernatremia and
angle at which the light beam enters the prism; thus hyponatremia
SG scale is calibrated in terms of the angles at which  Evaluation of the secretion of and renal response to
light passes through the specimen. ADH
 Advantages:  Normal values:
 Small volume of specimen (1-2 drops)  Serum osmolality: 275-300 mOsm
 Temperature corrections unnecessary  Urine osmolality: 50-1400 mOsm
(compensated between 15°C and 38°C)  Urine: serum osmolality = 1:1
 Easier to use; thus, faster results  Urine: serum osmolality, controlled fluid intake and
 Setbacks: injection of ADH = 3:1
 Correction for protein and glucose (same with the
urinometer) REAGENT STRIP
 Reading above 1.040 (usually seen in patient  Based on the change in pKa of polyelectrolyte in
recently injected with radiographic contrast media alkaline medium
or patients who are receiving plasma expanders)  Measures the ionic concentration (released hydrogen
should be diluted and measured again, or ions) of urine
measured by reagent strip or osmometry  Higher concentration = more hydrogen ions released
 Calibration: = lower pH
 Distilled water – 1.000  The pH indicator (bromthymol blue) on the reagent
 5% NaCl 1.022 +/– 0.001 pad measures the change in pH which relates to the
 9% sucrose – 1.034 +/– 0.001 specific gravity of the urine
 Blue (1.000/alkaline)  green  yellow (acid/1.030)
FALLING DROP METHOD  Advantage:
 More accurate than refractometer and more precise  No interference by large organic molecules (e.g.
than urinometer urea and glucose) as well as by radiographic
 Utilizes a column filled with water-immiscible oil contrast media and plasma expanders
where a measured drop of urine is introduced  More convenient: eliminated the need for additional
 As the drop of urine falls; it encounters two beams of procedure
light:  Setbacks:
 Breaking the first light: starts the timer  Only read at 0.005 intervals
 Breaking the second light: turns the timer off  pH 6.5 or higher have decreased readings caused
by interference with the indicator
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 High concentration of protein increases the 3. Hypersthenuria – SG >1.010
readings  Decreased fluid intake/water restriction
 Dehydration
Clinical Significance  Diarrhea
1. Monitoring patient hydration and dehydration  Diabetes mellitus/glycosuria
2. Loss of renal tubular concentrating ability  SIADH
3. Diabetes insipidus  Proteinuria
4. Determination of unsatisfactory specimens due to low  Lipid nephrosis
concentration  Fever
 Eclampsia
Laboratory Correlations  Radiographic contrast media
1. Isosthenuria – SG fixed at 1.010  Dextran administration
 Faulty tubular re-absorptive and secretory  CHF/heart failure
functions  Liver disease
2. Hyposthenuria – SG <1.010  Renal stenosis
 Polydipsia (increased fluid intake)  Adrenal insufficiency
 Intake of diuretics (natural/medicinal)
 Diabetes insipidus  Polyuria + hypersthenuria = diabetes mellitus
 Hypothermia  Polyuria + hyposthenuria = diabetes insipidus
 Protein malnutrition
 Hypertension ODOR
 Glomerulonephritis/pyelonephritis  Seldom of clinical significance and not part of routine
 Sickle cell anemia urinalysis
 Collagen disease

POSSIBLE CAUSES OF URINE ODOR

ODOR CAUSE
Faint, aromatic Normal odor; volatile aromatic acids
Foul / ammonia-like / fetid Urinary tract infections, bacterial proliferation
Distinctive / unusual / pungent Ingestion of onion, garlic, asparagus or thymol; smells like sulfur
Sweet / fruity Ketones (diabetes mellitus, starvation, vomiting or dehydration)
Maple syrup / caramel / burnt sugar Maple syrup urine disease
Mousy / musty Phenylketonuria
Rancid Tyrosinemia / tyrosinuria
Sweaty feet Isovaleric academia
Cabbage / hops Methionine malabsorption
Rotting fish Trimethylaminuria
Bleach Contamination
Lack of odor Acute tubular necrosis (ATN) in patients with acute renal failure

CHAPTER 5: CHEMICAL EXAMINATION OF URINE

REAGENT STRIPS How to Use Reagent Strips
 Provides a simple rapid means for performing 1. Dip the reagent strip briefly into a well-mixed
medically significant chemical analysis of urine. uncentrifuged urine specimen at room temperature.
 Packaged in opaque containers with desiccant inside 2. Remove excess urine by touching the edge of the strip
to protect them from light and moisture to the container as the strip is withdrawn. Blot the
 2 major types of reagent strip: Multistix, Chemstrip edge of the strip on a disposable absorbent pad.
3. Wait for 1min for the reaction to occur. Compare the
color reaction of the strip pads to the manufacturer’s
color chart in good lighting.
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PH this will form the ammonium ions (NH4–) which will then be
 The kidneys, together with the lungs are the primary excreted to the final urine. If there is an additional need for
elimination of hydrogen ions, the distal convoluted tubule (DCT)
regulators of the acid-base content of the body and collecting ducts can also produce ammonium ions.
These are the 3 processes that happen to regulate acid-
 How does the kidneys regulate acid-base balance? base balance. A disruption in this function can result in metabolic
The kidneys do this through the secretion of hydrogen in acidosis or renal tubular acidosis (inability to produce acid urine).
the form of ammonium ions, hydrogen phosphate, and weak
organic acid and by the reabsorption of bicarbonate from the
 No normal values are assigned to urine pH, but the
filtrate in the convoluted tubules.
To maintain the normal blood pH of 7.4, the blood following patterns are observed:
must buffer and eliminate the excess acid formed by dietary  Random: 4.5-8.0
intake and body metabolism. The buffering capacity of blood  First morning: 5.0-6.0 (acidic)
depends on HCO3–. Bicarbonate ions readily filter by the  Postprandial: alkaline
glomerulus should come back to the blood to maintain the proper
 Normal fresh urine cannot reach pH 9 (indicates old
pH. The reabsorption of bicarbonate must be 100% . This occurs
primarily in the proximal convoluted tubule (PCT). For the bacteria-contaminated urine)
bicarbonate to be reabsorbed, hydrogen ions are needed.  Diet and medication regulation
Hydrogen ions (H+) have small molecular size which are also  Meat = acid pH
readily filtered and absorbed and they are secreted by the renal  Vegetables = alkaline pH (except cranberry juice)
tubular cells into the filtrate then it will bind to HCO3– therefore,
 Medications for urinary tract infection (maintain an
it will not be excreted through urine, it will be filtered instead,
and will come back to plasma. acid pH)
If there is excess H+, the renal tubular cells will secrete  pH should be considered in conjunction with other
hydrogen ions into the filtrate. The ones that did not combined patient information including:
with bicarbonate ion will combine to the filtered phosphate ions  Blood pH
(HPO4–) and will then become dihydrogen phosphate (H 2PO4))
 Patient’s kidney status
which will be excreted into the final urine, so they are not
reabsorbed.  Presence of UTI
For the production of ammonia in PCT from the  Patient’s diet
breakdown of amino acid glutamine (NH3), there will be ammonia  Age of the specimen
which will go to the filtrate and will react to hydrogen ions and

CAUSES OF ACID URINE CAUSES OF ALKALINE URINE

“Sleeping” “Eating”
Emphysema Renal tubular acidosis
Systemic acidosis / diabetic ketoacidosis Systemic alkalosis / hyperventilation
Acidification therapy Alkalization therapy
Presence of acid-producing bacteria Presence of urease-producing bacteria
Starvation Vomiting
Dehydration Vegetarian diet
Diarrhea Old specimens
High-protein diet Detergent contamination
Cranberry juice
Medications – methenamine, mandelate, fosfomycin thromethamine

Clinical Significance  Prevention and control of renal calculi

1. Aid in establishing the presence of systemic acid-base precipitation/formation
disorders of metabolic or respiratory origin – Formed in acidic urine: calcium oxalate
 Respiratory/metabolic acidosis/ketosis not related – Formed in alkaline urine: calcium phosphate
to renal function disorders: acidic urine pH  Precipitation/identification of crystals
 Respiratory/metabolic alkalosis not related to renal  Treatment of urinary tract infections
function disorders: alkaline urine pH – UTI caused by urea-splitting organisms: maintain
 Defects in renal tubular secretion and reabsorption _____ urine
of acids and bases: renal tubular acidosis 3. Aid in excretion of specific substances
2. Management of urinary conditions that require the  Alkaline urine: salicylates
maintenance of a specific pH  Alkalization is induced with sodium bicarbonate,
potassium citrate and acetazolamide
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4. Determination of unsatisfactory specimens  Pre-renal proteinuria is not usually discovered in
routine urinalysis (reagent strip)
 What should be the pH of freshly excreted urine?  Examples of causes of pre-renal proteinuria:
 What causes the pH to be beyond the usual levels?  Intravascular hemolysis – hemolysis
 Muscle injury – myoglobin
Reagent strip: Double indicator
 Severe infection/inflammation – APRs
 Needed to measure between 5 and 9
 Multiple myeloma – Bence-Jones proteins
 Methyl red = 4-6 pH red/orange to yellow
 Bence Jones Protein
 Bromthymol blue = 6-9 pH green to blue
– Abnormal protein a.k.a. myeloma protein but not
 Interference: Only runover between acid from protein
specific for multiple myeloma
pad
– Low molecular weight serum protein
– Excessive amounts are seen in patients with
Methyl red + H+  Bromothymol blue – H+
( Red-orange  Yellow ) ( Green  Blue ) multiple myeloma
– Other diseases associated: leukemia, lymphoma,

PROTEIN macroglobulinemia, amyloidosis, other

 Protein determination is the most indicative of renal
malignancies
– Screening test: coagulates at 40-60°C and dissolves
disease
 Normal urine protein: <10 mg/dL or 100 mg/24 hours
at 100°C
– Uses heat and turbidity is measured
 Proteinuria is often associated with early renal
– Not all patients excrete detectable levels so the
disease
 Low molecular weight serum proteins are filtered;
suspected patient must be diagnosed by serum
many are reabsorbed electrophoresis or immunoelectrophoresis.
 Proteins found in normal urine:
 Albumin – primary protein of concern
RENAL PROTEINURIA
 Proteinuria associated with true renal disease which
 Serum and tubular microglobulins
 Tamm-Horsfall protein (uromodulin)
could be the result of either glomerular or tubular
 Proteins from prostatic, seminal and vaginal
damage
secretions
Glomerular Proteinuria
 Damage to glomerular membrane
Clinical Significance
 Selective filtration is impaired causing increased
 Clinical proteinuria is indicated at 30 mg/dL (300
mg/dL) or greater protein filtration (serum proteins, formed elements
 Demonstration of proteinuria does not always signify
such as RBCs and WBCs which can eventually be
renal disease excreted through urine)
 Amount of protein: up to 4 g/day
 Additional testing will determine if its presence
 Causes:
represents a normal or pathologic condition
 Abnormal substances deposit on the membrane
 Causes of proteinuria are grouped into three
– Primarily immune disorders result in immune
major or categories:
 Prerenal proteinuria
complex: lupus erythematous, streptococcal
 Renal proteinuria
glomerulonephritis
 Increased pressure on the filtration mechanism
 Postrenal proteinuria
(may be reversible)
– Hypertension, strenuous exercise,
PRERENAL PROTEINURIA
 Conditions affecting the plasma prior to reaching the
dehydration, pregnancy (preeclampsia),
kidneys orthostatic proteinuria
 Benign proteinuria (transient)
 Frequently transient and caused by increased levels
– Exposure to cold, exercise, dehydration, high
(exceeding the normal re-absorptive capacity) of low
molecular weight plasma proteins such as Hgb, fever
myoglobin and acute phase reactants (APRs)
Orthostatic (Postural) Proteinuria
 Postures that causes proteinuria

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 MEDT 15 ANALYSIS OF URINE AND BODY FLUIDS 18
 Benign proteinuria frequent in adults also measure creatinine to produce albumin-
 Possible causes: creatinine ratio
Exaggerated lordotic position which causes an  There is a simultaneous measurement of
unusual anatomical arrangement of the kidneys albumin and creatinine.
and their associated blood vessels that are  Provide an estimate of 24h albumin
compressed concentration from random urine
 Occurs in vertical position, disappears in  Albumin pad uses dye-binding reaction for
horizontal position specific albumin testing.
 Renal circulatory changes/renal congestion =  Albumin (Protein) Creatinine Ratio:
accumulation of blood in the kidney – Automated and manual methods available
 Testing procedures: – The results are in conventional and SI units
 Patient empties bladder before going to bed – Abnormal A:C ratio = 30-300 mg/g or 3.4-
 Patient collects first morning sample upon rising 33.9 mg/mol
 Patient collects a second sample after being in – Bayer Multistix Pro II strips measure
vertical position for several hours creatine, protein-high (traditional protein
method) and protein-low (new albumin-dye-
Tubular Proteinuria binding method)
 Tubular damage affecting re-absorptive ability so – Urobilinogen is not included on these strips
albumin and other low molecular weight proteins – Can be read manually or on instrumentation
will be present in the urine – Print-out is protein:creatinine ratio with
 Normally filtered albumin and other low molecular albumin result included on print-out
weight protein can no longer be re-absorbed – A chart is available for manual calculation
 Amount of protein: much lower levels – Specimens with a creatinine reading of 10
 Causes (Acute Tubular damage): are too dilute to interpret; recollect
 Exposure to toxic substances and heavy metals – Normal: 80 mg albumin/g creatinine or
 Severe viral infections <300 mg/protein/g creatinine
 Fanconi syndrome (generalized PCT defect) – Strip result of 15 mg/dL albumin is
indicative of clinical albuminuria
Microalbuminuria 2. Micral Test (Microalbumin Test):
 One of the first detectable signs of diabetic  Semi-quantitative reagent strip method
nephropathy which is also associated with  The reagent strips are read usually and first
increased risk of cardiovascular disease morning specimens are recommended
 Independent risk factor for renal mortality; more  Contains gold-labeled anti-human albumin
prevalent in hypertensive patients antibody-enzyme conjugate
 Indicator of early and possible reversible  Dip strip in urine to marked level for 5sec;
glomerular damage  Albumin binds to antibody
 Presence of albumin above normal levels but below  Bound and unbound conjugates move up strip
the detectable range of common urine dipstick  Unbound removed in captive zone containing
protein reagent pads albumin; bound continues up strip
 Testing:  Urine albumin bound conjugates:
– 24h urine specimen is required  Reaches enzyme substrate
– Tested using quantitative procedures for albumin  Colors will be produced from white (–) to
and reported in mg/24h of albumin red (varying degrees)
– Considered significant when 30-300 mg of  Compare color to chart
albumin are excreted in a 24h-period  Results read from 0-10 mg/dL
 Tests for Microalbumin 3. Immunodip Test (Microalbumin Test):
1. Reagent strip (Microalbumin Tests):  Uses an immunochromatographic technique
 Clinitek microalbumin reagent strips &  The strips are individually packaged and
Multistix Pro reagent strips specially designed container for strip
 These methods are based on immunochemical  Place container in controlled amount of
assays or albumin specific reagent strips that specimen for 3min, urine enters container
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 MEDT 15 ANALYSIS OF URINE AND BODY FLUIDS 19
 Albumin binds to blue latex particles coated 2. Sulfosalicylic acid (SSA) Confirmation
with anti-human albumin antibody  Not very common now due to instrumentation
 Bound and unbound migrate up strip which can  SSA precipitates proteins without heat
be controlled by the size of the particles,  Use centrifuged specimen
therefore the unbound particles do not  Compare against standards for 1+, 2+, 3+, 4+
migrate as far as the bound particles  Other substances precipitated: radiographic dye,
 Unbound encounters area of immobilized antibiotics, tolbutamide
albumin on strip which forms blue band 3. Regent Strip Reactions
 Bound continues migrating to an area of an  Protein error of indicators to produce a visible
immobilized antibody and forms second blue colorimetric reaction
band  Certain indicators change color in the presence of
 Top band = bound particles; bottom band = protein at a constant pH
unbound particles  Protein, primarily albumin, accepts H+ from the
 Color of band is compared with chart indicator
 Darker bottom band represents <1.2 mg/dL  Most sensitive to albumin because albumin has
albumin more amino groups to accept H+ than other
 Equal band colors = 1.2-1.8 mg/dL proteins
 Darker top band is 2.0-8.0 mg/dL albumin  Reagents: Tetrabromophenol blue (Multistix) or
tetrachlorphenol tetrabromosulfonphthalein
POSTRENAL PROTEINURIA (Chemstrip) and an acid buffer
 Proteins are added to urine as it passes through the  At pH level 3, both indicators are yellow
structures of the genitourinary tract  Color progresses through green to blue
 Causes:  Report: (–) trace 1+, 2+, 3+, 4+, or 30, 100, 300,
 Microbial infections (bacterial, fungal) causing 2000 mg/dL
inflammations and release of interstitial protein  Trace values are <30 mg/dL
 Presence of blood as a result of injury/trauma
 Menstrual contamination or vaginal secretions pH 3.0
 Prostatic fluids or semen – spermatozoa Indicator (H+) + Protein  Protein + H+
( Yellow ) Indicator – H+
( Green / Blue )
Tests for Albumin
1. Heat and Acetic Acid Test
 Reaction Interference
 Reference method
 Highly buffered alkaline urine overrides acid
 Principle: Urine is coagulated by heat and
buffer system
precipitated by (5-10%) acetic acid and the degree  Leaving reagent pad in urine too long removes
of turbidity produced is proportional to the amount buffer
of protein present  Highly pigmented urine
 Positive results:
 Quaternary ammonium compounds, detergents,
 1+ diffused cloud
antiseptics, chlorhexidine
 2+ granular cloud
 False-negatives for proteins other than albumin
 3+ distinct floccule
 False-positive trace from high SG
 4+ large floccule, dense, sometimes solid

TESTS FOR PROTEINS (Albumin)

1. Reagent strip + + –
2. Sulfosalicylic acid / cold precipitation test + – +
3. Heat and acetic acid test – reference method + – +
Interpretations: Proteinuria Albuminuria Globulins in urine

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 MEDT 15 ANALYSIS OF URINE AND BODY FLUIDS 20
GLUCOSE – Cystinosis
 Most frequently performed chemical analysis on urine  Osteomalacia
due to its value in the detection and monitoring of  Pregnancy
diabetes mellitus – Temporary lowering of renal threshold,
 Normal urine glucose: <15 mg/dL placental hormones block the action of insulin
 It does not usually appear in the urine until the
plasma concentration exceeds the renal threshold of Tests for Glucose
160-180 mg/dL 1. Reagent Strip Reactions
 Higher blood glucose = glucosuria  Glucose oxidase reaction specific for glucose
 Terminologies:  Principle: Double sequential enzyme reaction
 Melituria – presence of any sugar in urine  Reagents: Glucose oxidase, peroxidase, chromogen,
 Glycosuria – presence of any reducing sugar in buffer on test pad
urine  Report: (–) trace, 1+, 2+, 3+, 4+
 Glucosuria – presence of glucose in urine  100-2 mg/dL
 Non-glucose sugars seen in urine:  0.1-2%
 Galactose
 Fructose Glucose oxidase
 Pentose
Glucose + O2 (air)  gluconic acid + H2O
Peroxidase
 Lactose H2O2 + chromogen  oxidized colored chromogen +H2O
 Sucrose
 Reaction Interference
Clinical Significance  False (+) if there is peroxide or oxidizing
1. Glycosuria with hyperglycemia detergents
 Diabetes mellitus: Type 1, Type 2, GDM  False (–) if there is enzymatic reaction
 Pancreatic disease: interference
– Pancreatitis – Ascorbic acid and string reducing agents
– Pancreatic cancer – High levels of ketones (unlikely)
– Advanced stages of cystic fibrosis – High specific gravity and low temperature
 Endocrine disorders – Biggest error is old specimens due to
– Acromegaly glycolysis
– Cushing syndrome 2. Copper Reduction Test
– Hyperthyroidism/thyrotoxicosis  General test for glucose and other reducing sugars;
– pheochromocytoma non-specific
 Central nervous system damage  Rely on the ability of glucose and other substances
 Stress to reduce copper sulfate to cuprous oxide with
2. Renal glycosuria/glycosuria without hyperglycemia alkali and heat
 Appears when the reabsorption of glucose by the  A color change progressing form a (–) blue to
renal tubules is compromised green, yellow and orange or red or when the
 Kidney diseases: reaction takes place
– Fanconi syndrome
– Advanced renal disease

heat alkali
CuSO4 (cupric sulfate) + reducing substance  Cu2O (cuprous oxide) + oxidized substance  color (blue/green  orange/red)

 Clinitest – Reducing substance + CuSO4 = color

– Clinitest tablets: copper slfate, sodium – Clinitest Procedure:
carbonate, sodium citrate sodium hydroxide 1. Place a glass test tube in a rack, add 5 drops
– Sodium citrate + NaOH = heat of urine
– Sodium carbonate + CO2 blocks room air
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 MEDT 15 ANALYSIS OF URINE AND BODY FLUIDS 21
2. Add 20 drops of distilled water to the urine in levels, where the color goes from blue through
the test tube red back to green-brown). As a solution repeat
3. Drop one Clinitest tablet into the test tube and using the two-drop procedure: 10 drops of
observe the reaction until completion water, 2 drops of urine, values up to 5 g/L vs 2
(cessation of boiling) g/L
Caution: The reaction mixture gets very hot;  Reaction Interference
do not touch the bottom area of the test tube, – Not a specific test for glucose
use glass test tube only – Reducing substances
4. Wait for 15sec after boiling has stopped and  Galactose, lactose, fructose, maltose,
gently shake the contents of the tube pentoses, ascorbic acid, cephalosporins
5. Compare the color of the mixture to the – Major use is quick screen for galactosemia in
Clinitest color chart and record the result in newborns
mg/dL or percent  Newborn screening programs using blood are
6. Observe for the possibility of the “pass- being incorporated
through” phenomenon (caused by high glucose

TEST FOR SUGARS (Glucose)

1. Copper reduction tests
a. Classic Benedict’s + + –
b. Clinitest
2. Reagent strip – glucose oxidase – + –
Interpretations: Glycosuria Glycosuria other than glucose Small quantity of glucose or oxidizing

SUMMARY OF GLUCOSE OXIDASE AND CLINITEST REACTIONS

GLUCOSE OXIDASE CLINITEST INTERPRETATION
1+ (+) (–) Small amount of glucose present
4+ (+) (–) Possible oxidizing agent
(–) (+) Non-glucose reducing substance present; possible substance for reagent strip

KETONES Clinical Significance

 The term “ketones” refers to the three intermediate 1. Inability to metabolize carbohydrates
products formed during the catabolism of fatty acids  Primary utility: management and monitoring of
1. Acetones – 2%; formed irreversibly from diacetic insulin-dependent/Type 1 Diabetes mellitus
acid/acetoacetic acid; detected by reagent strip – Insulin dosage monitoring
2. Acetoacetic acid – 20%; first ketone formed from  Detection of diabetic acidosis/ketoacidosis
acetyl CoA; detected by reagent strip 2. Increased loss of carbohydrate
3. B-hydroxybutyric acid – 78%; formed reversibly  Vomiting
from diacetic acid/acetoacetic acid  Diarrhea
 Does not normally appear in urine as metabolized fats  Strenuous exercise
completely broken down to carbon dioxide and water 3. Inadequate intake of carbohydrate
 Appear only in urine when use of carbohydrates as  Starvation/fasting/eating disorders
source of energy becomes compromised (fats are  Malabsorption/pancreatic disorders/malnutrition
broken down to glucose for energy) 4. Inborn errors of metabolism
 Normal blood ketones: 2-4 mg/dL
 Accumulation of ketones in the blood leads to Test for Ketones:
electrolyte imbalance, dehydration, acidosis, diabetic 1. Reagent Strip Reactions
coma  Measure primarily acetoacetic acid
 Testing for urinary ketones is most valuable in the  Slightly sensitive to acetone in presence of
management and monitoring of insulin dependent DM glycine (Chemstrips)
(Type I DM)  Primary reagent: sodium nitroprusside
(nitroferricyanide)
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alkali 4.Compare the tablet color with the manufacturer-
acetoacetic acid + nitroprusside  purple supplied color chart
5. Report: (–), small, moderate, or large
 Report: (–), small (1+), moderate (2+), large (3+),
 Reaction Interference
or small (5, 15), moderate (40), large (80, 160)  Phthalein dyes in medication, red-colored urine
mg/dL  Medications with sulfhydryl groups
2. Acetest  False (+) from too long timing
 Not a confirmatory test
 False (–) from too old specimens
 Can perform serial dilutions (provides more
– Volatilization and bacterial breakdown of
information on the extent of ketosis) and use serum acetoacetic acid
 Useful if the urine has an interfering color
 Tablet = sodium nitroprusside, glycine, disodium
BLOOD
phosphate, lactose (gives better color); 10x more  Blood may be present in urine in the form of intact
sensitive to diacetic acid RBCs or as the product of RBC degradation,
hemoglobin
Acetoacetic acid (acetone) + sodium nitroprusside +
 Normal urine blood count: <5 RBC/µL
glycine  purple
 Abnormal blood levels in urine is most accurately

 Acetest Procedure:
detected by the chemical test for hemoglobin,
1. Remove the Acetest tablet from the bottle and however, a positive reagent strip testing indicates
place on a clean, dry piece of white paper either hematuria, hemoglobinuria or myoglobinuria
2. Place one drop of urine on top of the tablet which signify different clinical importance
 The reagent strip for blood is more accurate then
3. Wait for 30sec
microscopic for detecting blood

CLINICAL SIGNIFICANCE AND DIFFERENTION

CLINICAL SIGNIFICANCE
HEMATURIA HEMOGLOBINURIA MYOGLOBINURIA
Normal urine, cloudy red Clear, pink/red/red-brown urine Heme containing protein in
urine Intravascular hemolysis muscle tissue
Renal or urogenital tract  Hemolytic anemia Clear, red/brown urine
 Calculi or tumors  Transfusion reactions Rhabdomyolysis/muscle
 Glomerulonephritis  Severe burns destruction
 Pyelonephritis  Infections/malaria  Muscular trauma/crush
 Trauma  Strenuous exercise syndromes
 Exposure to toxic chemicals  RBC trauma  Muscle-wasting disease
 Anticoagulant therapy  Brown recluse spider bites  Alcohol/drug overdose
 Extensive exertion
 Convulsions
Physiological Lysis of blood in urine
 Prolonged coma
 Strenuous exercise  Happens _____ urine
 Side effects of cholesterol-
 Menstrual contamination  Mixture of hematuria and hemoglobinuria
lowering statin
medications
Toxicity to kidneys Toxicity to kidneys Toxicity to kidneys
URINE FINDINGS
Macro

Chem/Micro
Renal problems:  Protein: _____
 Protein: _____  RBC: _____
 RBC: _____  Occasional pigment casts  Protein: _____
 Hemosiderin: yellow-brown granules  RBC: _____
Urogenital tract problems: – 2-3 days after an acute hemolytic episode  Dense brown casts
 Protein: _____ – Hemochromatosis / true siderosis of the
 RBC: _____ kidney parenchyma
PLASMA FINDINGS
 Normal plasma color  Pink plasma  Normal plasma color
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 MEDT 15 ANALYSIS OF URINE AND BODY FLUIDS 23
 Normal haptoglobin  Low haptoglobin  Normal haptoglobin
 Increased enzymes
Reagent strip test: all conditions are positive

HEMOGLOBINURIA MYOGLOBINURIA
 Clear, red urine
 Clear, red urine
 Toxic to renal tubules
 Toxic to renal tubules
 Clear plasma
 Red plasma
 Increased CK enzymes
 Urine separation: Hemoglobin + NH4SO4 = clear supernatant
 Urine separation: Myoglobin + NH4SO4 = red supernatant

Other Tests to Differentiate Hemoglobin and  RBCs  RE system  protoporphyrin  bilirubin

Myoglobin: (unconjugated bound to albumin) circulates to liver 
1. Blondheim’s Test/Ammonium Sulfate Method conjugated bilirubin  bile duct  intestine 
 2.8g ammonium sulfate + 5mL centrifuged urine is stercobilinogen + urobilinogen
mixed until dissolved. Sit for 5 minutes, then filter – Bilirubin are from RBCs.
or centrifuge. – If under normal conditions, when 120 days have
 Hemoglobin = clear supernatant, (–) blood passed, RBCs are destroyed in the spleen and liver
 Myoglobin = red supernatant, (+) blood by the phagocytic cells of reticuloendothelial
2. Absorption Spectrophotometry system (RE system).
3. Electrophoresis – Hemoglobin will be broken down into components
4. Immunoassays (iron, protein, protoporphyrin).
– The body will reuse iron and protein and the cells
Test for Blood (in general): of RE system will convert protoporhyrin to bilirubin
1. Reagent Strip Reactions so unconjugated bilirubin will therefore be
 Principle: pseudoperoxidase activity of hemoglobin produced. This will be released into the circulation
and eventually it will bind to albumin and will be
hemoglobin transported to liver. The unconjugated bilirubin will
H2O2 + chromogen  oxidized chromogen + H2O not be excreted through urine because it is bound
peroxidase to albumin and it is water-insoluble.
– In the liver, the unconjugated bilirubin is
 Intact RBCs show a speckled pattern
conjugated with glucuronic acid by the action of
 Report: trace, small (1+), moderate (2+), large
glucuronyl transferase to form water-soluble
(3+) bilirubin, diglucoronide or conjugated bilirubin.
 Sensitivity 5 RBCs/µL
– Usually this, conjugated bilirubin does not appear
 Reaction Interference:
in the urine as well, because it is passed directly
 False (+): strong oxodozing agents, bacterial from the liver into the bile duct to the intestine.
peroxidases, menstrual contaminiation – When the conjugated bilirubin is excreted through
 False (–): high SG/crenated cells the bile duct into the intestine, the intestinal
 Formalin bacteria will convert bilirubin to a combination of
 Captopril stercobilinogen and urobilinogen.
 High concentration of nitrite  Stercobilinogen  stercobilin  feces
 Ascorbic acid >25 mg/dL – Stercobilinogen will not be reabsorbed and will only
 Unmixed specimens stay in the intestine where it will oxidize into
stercobilin which will be seen in the feces
BILIRUBIN  Urobilinogen  blood  liver (passes through
 Highly pigmented yellow compound which is the
kidney and some is excreted)
degradation product of hemoglobin – Urobilinogen will not be reabsorbed and will only
 Can provide an early indication of liver disease before
stay in the intestine where it will oxidize into
a patient exhibit jaundice urobilin which will be seen in the feces
 Its presence or absence in urine may also be used to
– Some urobilinogen are reabsorbed from the
differentiate the cause of jaundice intestines to the blood and will circulate in the liver
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 MEDT 15 ANALYSIS OF URINE AND BODY FLUIDS 24
then will be excreted back into the intestine 3.Place one drop of water onto the tablet, and wait
through the bile duct and the cycle goes on 5sec
4. Place a second drop of water onto the tablet so
Clinical Significance that the water runs off the tablet onto the mat
1. Conjugated bilirubin appears in urine with bile duct 5. Observe the color of the mat around the tablet at
obstruction and liver disease the end of 60sec. The presence of a blue-to-
2. Obstruction: bilirubin backs up into circulation and is purple color on the mat indicates that bilirubin is
excreted in urine; no urobilinogen is formed present; a slight pink or red color should be
3. Hepatitis, cirrhosis: conjugated bilirubin leaks back ignored; report as (+) or (–).
into circulation from damaged liver; some bilirubin  Reaction Interference:
passes to intestine  False (+): urine pigments, pyridium, indicant,
4. Hemolytic disease: increased unconjugated bilirubin Lodine
= jaundice; no urine bilirubin, increased urobilinogen  False (–): old specimens (biliverdin does not
5. Hepatic jaundice react), high ascorbic acid and nitrite because the
 Hepatitis combine with diazonium salt and block bilirubin
 Cirrhosis reaction
 Other liver disorders
6. Post-hepatic jaundice UROBILINOGEN
 Bile duct obstruction: gallstones, tumors  Bile pigment produced in the intestine
 Cholestasis  Produced together with stercobilinogen when
conjugated bilirubin is excreted through bile duct into
Tests for bilirubin: the intestine
1. Reagent Strip Reactions  Urobilinogen may be reabsorbed to the bloodstream
 Principle: Diazo reaction while stercobilinogen is not
 Excreted in small amounts in urine: <1 mg/dL or 1
acid Ehrlich unit
bilirubin glucuronide + diazonium salt  azodye  Can provide an early indication of liver disease
tan shades or pink to violett
 Specimen collection is between 2-4 pm

 Report: (–), small (1+), moderate (2+), large (3+)

Clinical Significance
1. Early detection of liver disease
 Colors may be difficult to interpret
2. Liver disorders, hepatitis, cirrhosis, carcinoma
 Atypical colors can be a problem for automated
3. Hemolytic disorders: excess bilirubin being converted
readers
to urobilinogen and high urobilinogen recirculated to
2. Ictotest Tablets liver
4. (–) bilirubin and strong (+) urobilinogen are seen in
 Tablet test based on the same diazo reaction as the
dipsticks but is less subject to interferences and hemolytic disorders
5. No urobilinogen is seen in the urine with bile duct
much more sensitive
 Confirmatory test for bilirubin
obstruction; strip will give a normal result
6. There cannot be a (–) urobilinogen reading from
 More sensitive: 0.05-0.1mg/dL
 Reagent strips: 0.40 mg/dL
reagent strip
7. Pre-hepatic jaundice
 Ictotest may be requested for early disease
 Hemolytic disorders
 Use specified mat for test; mat keeps bilirubin on
8. Hepatic jaundice
surface for reaction
 Hepatitis
 Ictotest Procedure:
 Cirrhosis
1. Place 10 drops of urine onto one square of the
 Other liver disorders
absorbent test mat
9. Post-hepatic jaundice
2. Using forceps, remove one Ictotest reagent
 Bile duct obstruction: gallstones, tumor
tablet, recap the bottle promptly, and place one
 cholestasis
drop of water onto the tablet in the center of the
10. Constipation: 1% of non-hospitalized patients, 9%
moistened area
hospitalized patients
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 MEDT 15 ANALYSIS OF URINE AND BODY FLUIDS 25
Tests for urobilinogen: 2. Ehrlich’s Tube test
1. Reagent Strip Reactions  Differentiation among urobilinogen,
 Based on the Ehrlich aldehyde reaction porphobilinogen, and Ehrlich reactives
 Different principles for Multistix and Chemstrip  Addition of Ehrlich reagen to urine produces a
 Multistix: p-dimethylaminobenzaldehyde (Erlich cherry red color enhanced by sodium acetate
reagent)  Test is modified to differentiate urobilinogen,
– Report in Ehrlich units (EU)  1 EU = 1 mg/dL porphobilinogen, and Ehrlich reactive compounds
– Normal readings 0.2-1 EU, abnormal 2, 4, 8  Non-specific test; it does not actually specify if
 Chemstrip: 4-methoxybenzen-diazonium- urobilinogen, porphobilinogen or Ehrlich reactive
tetrafluoroborate compound is present so this is modified by addition
– Diazo reaction of chloroform and butanol
– More specific than Ehrlich reaction  To determine urobilinogen, porphobilinogen, and
– Report in mg/dL excess urobilinogen
 A true absence of urobilinogen is not detectable  Tubes with red color
 Reaction Interference:  Add chloroform, shake
 Ehrlich reactive compounds: porphobilinogen,  Add butanol to second tube, shake
indican, sulfonamides, methyldopa, procaine,  If urobilinogen it will be extracted into both
chlorpromazine, p-aminosalicylic acid because it is soluble in both chloroform and
 Both result: urobilinogen is highest after meals butanol
(increased bile salts), old specimens and formalin  Porphobilinogen extracted into none
preservation decrease results  Reactives extracted into butanol
 Chemstrip: false (–) with high nitrite interferes
with diazo reaction

UROBILINOGEN PORPHOBILINOGEN EHRLICH-REACTIVE UROBILINOGEN/ PORPHOBILINOGEN EXCESS UROBILINOGEN

Tube 1 Tube 2 Tube 1 Tube 2 Tube 1 Tube 2 Tube 1 Tube 2 Tube 1 Tube 2
UR B UR B UR B UR B UR B
C UR C UR C UR C UR C UR

3. Watson-Schwartz Differentiation Test  Immediate red color on top and throughout when
4. Hoesch Screening Test shaken
 For acute porphyrias (chapter 9)  Rapid screening test for monitoring of urinary
 Hoesch reagent: Ehrlich reagent in 6 M Hcl porphobilinogen
 Two drops urine in 2 mL Hoesch reagent  High pH inhibits urobilinogen
 False (+): methyldopa, indican, pigmented urine

BILIRUBIN AND UROBILINOGEN FINDINGS IN JAUNDICE

URINE BILIRUBIN URINE UROBILINOGEN FECAL COLOR
Hemolytic Disease – +++ dark
Liver Damage + or – ++ pale/dark
Bile Duct Obstruction +++ – pale

NITRATE 2. Contact time of bacteria with the urinary nitrate

 Rapid screening test for the presence of urinary tract 3. Presence of adequate amounts of urinary nitrate
infection or asymptomatic bacteriuria 4. Other factors:
 Not intended to replace urine culture as the primary  Antibiotic therapy
test for diagnosis and monitoring of bacterial  Large quantities of ascorbic acid
infections  High specific gravity

Factors that Influence Nitrate Test Results Clinical Significance

1. Presence or absence of the nitrate reductase enzyme 1. Cystitis (bladder)
in the infectious agent 2. Pyelonephritis (tubules)

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3. Evaluation of antibiotic therapy – Insufficient contact time between bacteria and
4. Monitoring patients at high risk of UTI urinary nitrate
5. Screening urine culture specimens (in combination – Lack of urinary nitrate
with LE test): DM, pregnancy, recurrence – Large quantities of bacteria converting nitrite
to nitrogen
Test for Nitrate – Presence of antibiotics
1. Reagent Strip Reaction – High concentrations of ascorbic acid
 Tests ability of bacteria to reduce nitrate to nitrite – High specific gravity
 Principle: Greiss reaction: nitrite reacts with  False (+):
aromatic amine to form a diazonium salt that then – Old specimens (bacterial multiplication)
reacts with tetrahydrobenzoquinoline to form a – Highly pigmented urine
pink azodye – Pink edges or spotting on reagent strip is
 Sensitive for 100,000 organisms/mL considered (–)
 Results: (–) and (+) – Check automated readers manually for color
 Reaction Interference: interference
 False (–):
– Non-reductase-containing bacteria

acid
para-arsanilic acid or sulfanilamide + NO2  diazonium salt
acid
diazonium salt + tetrahydrobenzoquinoline  pink azodyee

LEUKOCYTE ESTERASE
 Detects the presence of esterase granulocytes and Test for Nitrate
monocytes but does not detect lymphocytes 1. Reagent Strip Reaction
 Normal urine leukocytes: 0-5/HPF  LE catalyzes hydrolysis of acid esterase on pad to
 Due to vaginal contamination women tend to have aromatic compound and acid
higher numbers than men  Aromatic compounds reacts with diazonium salt on
 Not considered a quantitative test; do microscopic if pad for purple color
(+)  Reaction Interference:
 Advantage: detects presence of lysed leukocyte  False (+):
– Strong oxidizing agents
Clinical Significance – Formalin
1. Bacterial urinary tract infection – Highly pigmented urine, nitrofurantoin
2. Non-bacterial urinary tract infection: Trichomonas,  False (–):
Chlamydia, yeast – High concentrations of protein, glucose, oxalic
3. Inflammation of the kidneys and the urinary tract acid, ascorbic acid, gentamicin,
4. Screening of the urine culture specimens in cephalosporins, tetracyclines
conjunction with nitrite but a better predictor than – Crenation from high SG
nitrite – Inaccurate timing (must have 2min)
5. Interstitial nephritis

leukocyte acid
indoxylcarbonic acid ester  indoxyl + acid indoxyl + diazonium salt  purple azodyel
esterases

+
SPECIFIC GRAVITY  High concentration = more H released
 Based on pKa (dissociation constant) of  Indicator bromthymol blue measures pH change
apolyelectrolyte in alkaline medium  Blue (alkaline) through green to yellow (acid)
+
 Principle: Polyelectrolyte ionizes releasing H in
relation to concentration of urine

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Reaction Interference  Slight elevation from protein
 No interference from large molecules, glucose and  Decreased readings: urine pH 6.5 or higher
urea and radiographic dye and plasma expanders – Interferes with indicator; add 0.005 to the reading;
– Reason for difference in refractometer reading readers automatically add this

TEST (CORRELATE) PRINCIPLE/SENSITIVITY REAGENT FALSE-POSITIVE FALSE-NEGATIVE

Double-indicator system
M: 5.0-8.5 Methyl red  Run-over from adjacent pad
 Old unpreserved specimen
C: 5.0-9.0 Bromthymol blue (protein)
pH (nitrite, LE,
*0.5 Intervals
micro)
REACTION/COLOR: methyl red + H+  bromthymol blue – H+
yellow  green  deep blue
pH 5.0  9.0
 Highly buffered alkaline
urine
M: Tetrabromophenol  High S.G.
Protein (Sorensen’s)
blue  QUATS (detergent)  Microalbuminuria
error of indicators
C: Tetrachlorphenol  Antiseptics/ chlorhexidine  Proteins other than
M: 15-30 mg/dL
or Tetrabromo-  Prolonged exposure of the  albumin
Protein (blood, C: 6 mg/dL
sulfophthalein strip to reagent
nitrite, LE, micro)
 Pigmented specimens,
phenazopyridine
REACTION/COLOR: pH 3.0
indicator + protein  protein + H+
indicator – H+
yellow  blue-green
 High SG
M: Glucose oxidase,
Double sequential  High levels of ascorbic acid
peroxidase, KI2
enzyme reaction  Oxidizing agents and or ketones
C: Glucose oxidase,
M: 75-125 mg/dL detergents  Low temperature
peroxidase,
Glucose C: 40 mg/dL  Improper urine
tetramethylbenzidine
(ketones, proteins)  Preservation
Reaction/Color: glucose + O2 [glucose oxidase]  gluconic acid +H2O2
H2O2 + chromogen [peroxidase]  oxidized chromogen + H2O
M: green  brown
C: yellow  green
Sodium nitroprusside/  Highly pigmented urine
ferricyanide reaction  Phthalein dyes
Sodium nitroprusside  Improper urine
Ketones M: 5-10mg/dL AAA  Levodopa
C: with Glycine  Preservation
(glucose) C: 9 mg/dL AAA  Medications with free
70 mg/dL acetone sulfhydryl groups
Reaction/Color: acetoacetate (and acetone) + sodium nitroprusside + (glycine) [alkaline]  purple color
 Unmixed specimens
M: diisopropyl-
 High levels of ascorbic acid
benzene dehydro-  Strong oxidizing agents
 Captopril
peroxide tetra-  Vegetable and bacterial
 High specific gravity/
Pseudoperoxidase methylbenzidine peroxidases
Blood crenated cells
activity of hemoglobin C: dimethyl-  Menstrual contamination
(protein)  Formalin
dihydroperoxide  Povidoneiodine
 High concentration of nitrite
tetramethyl-  (Betadine)
(>10mg/dL)
benzidine
 High protein
Reaction/Color: H2O2 + chromogen [Hgb peroxidase]  oxidized chromogen (green-blue) + H2O
 Highly pigmented urines
M: 2,4-dichloroaniline  Ascorbic acid (>25mg/dL)
 Phenazopyridine
diazonium salt  Specimen exposure to
 Indican
Bilirubin Diazo reaction C: 2,6-dichloro- temperature and light
 Intestinal disorders
(urobilinogen) benzine diazonium  Highly concentrated
 Metabolites of Lodine
tetrafluoroborate  Nitrite
 Doses of chlorpromazine
Reaction/Color: bilirubin glucuronide + diazonium salt [acid]  azodye (tan, pink or violet)

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M: porphobilinogen
Indican
p-aminosalicylic acid M: old specimens
Sulfonamides Preservation in formalin
M: paradimethyl-
M: Erlich’s aldehyde p-aminobenzoic acid
aminoenzaldehyde
reaction Methyldopa C: old specimens
C: 4-methoxy-
C: Azo-coupling (diazo) Procaine Preservation in formalin
Urobilinogen benzene-diazo-
reaction Chlorpromazine High concentration of nitrite
(bilirubin) niumtetrafluroborate
Highly pigmented urine Patients receiving broad
spectrum antibiotics
C: highly pigmented urine
Phenazopyridine
Reaction/Color:
M: urobilinogen (Ehrlich’s reactive substance) + p-dimethylaminobenzaldehyde (Ehrlich’s reagent) [acid]  light pink to red
C: urobilinogen + diazonium salt [acid]  white to red azodye
Nonreductase-containing
bacteria
Insufficient contact between
bacteria and urinary nitrate
M: p-arsanilic acid
Lack of urinary nitrite
Tetra-hydrobenzo
Improperly preserved Large quantities of bacteria
(h)-quinolin-3-ol
Griess’ reaction specimens converting nitrite to nitrogen
Nitrite C: sulfanilamide, 3-
Highly pigmented urine Antibiotics
(protein) hydroxy-tetra-
Ascorbic acid
hydrodenzoquinoline
High specific gravity
Abnormally high levels of
urobilinogen
Acidic urine
Reaction/Color: p-arsanilic acid or sulfanilamide + nitrite [acid]  diazonium salt
diazonium salt + tetrahydrobenzoquinolin [acid]  pink azodye
Oxalic acid
Strong oxidizing agents Ascorbic acid
M: derivatized
Formalin High specific gravity
pyerole amino acid
Highly pigmented urine Protein (>500mg/dL)
Granulocytic ester, diazonium salt
Leukocyte Nitrofurantoin Glucose (>3g/dL)
esterase reaction C: indoxylcarbonic
(protein) Vaginal discharge Gentamicin
acid ester, diazonium
Imipenem Cephalosporins
salt
Clavulanic acid Tetracyclins
Inacurate timing
Reaction/Color: indoxylcarbonic acid ester [LE]  indoxyl + acid indoxyl + diazonium salt [acid]  purple azodye
M: poly (methyl) vinyl
ether/maleic
PK change of
anhydride)
polyelectrolyte in an High concentrations of Highly alkaline
Specific bromthymol blue
alkaline medium Proteins urines (pH >6.5)
gravity C: ethyleneglycol-Bis
nsahhasdsa 
(amino-ethylether)
bromthymol blue
Reaction/Color: blue  green  yellow
11 reagent pad Phosphomolybdate
*Ascorbic acid
Reaction/Color: ascorbic acid (>5mg/dL) + phosphomolybdate  molybdenum blue

CHAPTER 6: MICROSCOPIC EXAMINATION OF URINE

INTRODUCTION  White blood cells (WBCs)
 Microscopic examination is done to identify insoluble  Epithelial cells
substances/formed elements  Casts
 Red blood cells (RBCs)  Baceteria

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 Yeast parasites – Although refrigeration is a preservation method, it
 Mucus is not recommended
 Spermatozoa – Specimens from refrigeration needs to be warmed
 Crystals to 37°C before testing, in the process some crystals
 Artifacts can be dissolved
 Commonly used: Midstream clean-catch specimen
 What contributes to the presence of formed elements? – Less contamination
Blood, kidney, genitourinary tract, and external contamination. – Dilute random specimens cause false (–) to physical

 Does the presence of formed elements out-rightly

analysis
signify diseased conditions?  When the urine has been acquired, mix it before
decanting to the centrifuge tube
MACROSCOPIC SCREENING/CHEMICAL SIEVING
 Done to maximize time. How? Sediment Preparation
 Performed based on physical and chemical results  Specimen Volume
 Parameters: color, clarity, blood, protein, nitrite,  From the urine container, decant urine to the
leukocyte esterase, and possibly glucose centrifuge/conical tube up to the 10-15 mL mark
 Special populations that do not need screening: – Conical tubes are better than urine container
pregnant women, pediatric, geriatric, diabetic, because reagent strips are easily immersed in it
immunocompromised, renal patients – Reagent strips fit into 12 mL
 Always cap tubes for centrifugation
MACROSCOPIC SCREENING CORRELATIONS  Too little volume = fewer formed elements
SCREENING TEST SIGNIFICANCE – Specimen volume should be noted if a 10-15 mL
Color Blood urine is not obtained (pediatric patients)
Hematuria vs Hemoglobinuria/
 Some laboratories correct for volume
Myoglobinuria
Clarity – Ex. Only 6 mL of urine was obtained, the result
Confirm pathologic or non-pathologic
cause of turbidity will be multiplied by 2 (depends on the lab)
Blood RBCs/ RBC casts  Centrifugation
Protein Casts/Cells  Standard speed and time of centrifugation: 5min at
Nitrite Bacteria/WBCs 400 retrieve centrifugal force (RCF)
Leukocyte esterase WBCs/WBC casts/Bacteria
 RCF corrects for variations in the diameter of
Glucose Yeast
centrifuge heads; revolutions per minute does not
CLINICAL AND LABORATORY STANDARDS break the centrifuge
 Post-centrifuge sediment
INSTITUTE (CLSI)
 Pour urine onto the sink
 Provide and guidelines for medical professionals
 0.5-1.0 mL of urine sediment should be left after
through its unique consensus process
 Recommends to perform microscopic examination
decantation
 Concentration factor: volume of urine centrifuged/
when:
 Requested by a physician
sediment volume
– Probability of detecting low quantities of formed
 Laboratory-specified population
 Any abnormal physical or chemical result
elements
 Aspirate using pipette, rather than pour off urine
 Mix sediment gently, not vigorously
SEDIMENT STANDARDIZATION
 Least standardized, most time consuming and subject
to variations Sediment Volume
 Be consistent
 Commercial systems control this
Specimen Preparation
 Glass slide method:
 Examine when fresh or adequately preserved
 20 µL
– If not RBCs, WBCs, and casts will be lysed in dilute,
 Beyond will cause overflow when coverslip is on
alkaline urine
 Refrigeration precipitates crystals

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 Overflow will cause heavier elements (casts) to  Reduced light is essential
flow outside the examination area of coverslip,  Magnification is 10x and 40x
thus, will not be observed under microscope  Par focal means minimal adjustment when changing
 22x22 glass coverslip objectives (use fine adjustment)
 Lower light using the rheostat
Sediment Examination  Condenser can be raised up and down
 Be consistent  Do not use the aperture diaphragm
 Minimum 10 low power fields (lpf) and 10 high power 2. Phase-contrast microscopy
fields (hpf)  Increases refractive index of the sediment
 Low power: casts, general composition  Has halo effect around the element which helps for
– Scan edges for casts with glass slide method better imagery enforcement esp. in hyaline casts
 High power: identification of other formed elements 3. Polarizing microscopy
unable to read at lpf  Aides in the identification of crystals and lipids
 Initial focusing: low power, reduced light (droplets, oval fat bodies, fatty casts, cholesterol)
– Focus on epithelial cell, not artifacts that are in a – Crystals are multicolored
different plane – Cholesterol produces Maltese cross formations
 Use fine adjustment continuously for best view  Ability to split light into two beams
4. Interference-contrast microscopy
Reporting of Results  The object appears bright against dark background
 Consistent within laboratory and gives 3D images
– Rare, few, moderate, many, or full field or 1+, etc. 5. Dark-field and fluorescence microscopy
(semi-quantitative)  Dark-field Microscopy – identification of
 Casts: reported average per lpf Treponema pallidum
 RBCs, WBCs: reported average per hpf  Fluorescence Microscopy – visualization of
 Epithelial cells, crystals, etc.: reported in semi- fluorescent organisms
quantitative terms
URINE SEDIMENT CONSTITUENTS
SEDIMENT STAINS  Many urine have a rare epithelial cell
 Increase the overall visibility of sediment elements  Small amounts of constituents can be normal or
when using bright field microscopy pathogenic based on the clinical picture
 Identifies hyaline casts, nuclei, cytoplasm, and  Some constituents are easily distorted
inclusions on cells
 Sternheimer-Malbin (SHM) – consists of crystal Cellular Constituents
violet and safranin O, frequently used in UA 1. Red Blood Cells
 Acetic acid – enhances WBC nuclei 2. White Blood Cells
 Toluidine blue – enhances WBC nuclei 3. Epithelial Cells
 Lipid stains – uses oil red O and Sudan III to
confirm the presence of neutral fats, triglycerides RED BLOOD CELLS
and cholesterol (polarized, not stained)  Smooth, non-nucleated, biconcave discs
 Gram stain – identifies bacteria/bacterial casts  Shrunken and crenated: hypertonic urine
 Hansel stain – consists of methylene blue and  Swollen/ghost cells/shadow cells: hypotonic urine
eosin Y, used to identify eosinophils  Identified using HPO
 Prussian blue stain – confirms the presence of  Frequently confused with yeast, oil droplets, air
hemosiderin bubbles, starch
 Eosin – stains the RBC and helps differentiate it  Dysmorhic: RBCs that vary in size, have cellular
from yeast protrusions or are fragmented
 Iodine – stains the starch and vegetable fibers – Primarily associated with glomerular bleeding
– Physiologic: strenuous exercise
MICROSCOPY – Acanthocytic, blebs
1. Bright-field – Fragmented, hypochromic
 Most common in UA – Aid in diagnosis

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 Clinical Significance EPITHELIAL CELLS
 Normal value: 0-3 or 5/hpf  Inflammations Proteins are added to urine as it
 Increased RBCs in urine results from: passes through the structures of the GUT
– Damage to glomerular membrane  Types and Classification
(glomerulonephritis) a. Squamous – male and female urethra, vagina;
– Vascular injury in the GUT biggest epithelial cell
– Renal calculi b. Transitional (urothelial) – bladder, renal pelvis,
 Number of cells = extent of damage calyces, ureters, upper male urethra; irreg nucleus
 Macroscopic vs microscopic hematuria c. Renal Tubular Epithelial (RTE)
– Cloudy, red to brown urine: advanced disease,  Round nucleus
trauma, acute infection, coagulation disorders  Size and shape vary with renal tubular area
– Clear urine: early glomerular disease,  Proximal Convoluted Tubule (PCT) – larger,
malignancy, renal calculi confirmation rectangular (columnar)
 Non-pathologic:  Distal Convoluted Tubule (DCT) – smaller,
– Strenuous exercise/activity: seen with rounded/oval
hyaline/RBC/granular casts  Collecting ducts – cuboidal and never round
– Menstruation: contamination  Renal fragment – three or more cuboidal cells
 Clinical significance:
WHITE BLOOD CELLS  Clue cells
 Neutrophil is predominant  Squamous cell with pathologic significance
 Identified under HPO  Vaginal infection with Gardianella vaginalis
 Much easier to identify than RBCs due to their  Seen in urine but more common in vaginal
granules and multi-lobe nuclei, appear larger than specimens
RBCs but smaller than epithelial cells  Increased transitional epithelial cells (TEC):
 Glitter cells appearance show in: – Catheterization: TEC with normal morphology
 Hypotonic urine – Malignancy or viral infection: TEC with abnormal
 Brownian movement morphology
 Swell; granules sparkle  RTE cells: most clinically significant urine; indicate
 Pale blue of stained tubular necrosis; fragments indicate severe
 Non-pathologic destruction
 Clinical Significance: – Secondary effects of glomerular disorders
 Normal value: <5/hpf, more in females – Toxicity: heavy metals, drug-induced,
 May enter through glomerulus or trauma but also hemoglobin, myoglobin (physical exam: red
by amoeboid migration color)
 Increased WBCs in urine (pyuria) results from: – Infections: viral infections, pyelonephritis (+ LE,
– Infections: cystitis, pyelonephritis, prostatitis, nitrite, bacteria)
urethritis – Others: allergic reactions, malignancy, transplant
– Inflammations: glomerulonephritis, lupus rejection, salicylate poisoning (single cuboidal
erythematosus, interstitial nephritis, tumors cells)
 Increase in eosinophils results from:  Oval fat bodies (OFB)
– Drug-induced interstitial nephritis – RTE cells that have absorbed lipids
– Renal transplant rejection – Seen in conjunction with free-floating refractile
 Mononuclear cells (monocytes, macrophages, droplets
histiocytes) – Increased lipid in urine (lipiduria): nephrotic
– Lymphocytes, monocytes, macrophages, and syndrome, ATN, diabetes, crush syndrome
histiocytes are rare – Lipid-storage diseases: cause the presence of
– >30% increase – chronic inflammation large fat, laden histiocytes
– Differentiate form renal tubular epithelial (RTE) – Bubble cell: RTE cells containing large, nonlipid
cells vacuoles seen in cases of ATN; represent injured
– Lymphocytes may resemble RBCs; seen in renal
transplant rejection
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cells in which the ER has dilated prior to cell Casts
death  Elements unique to the kidneys
 Formed within the lumens of DCT and collecting duct
Miscellaneous Constituents providing a microscopic view within the nephron
1. Spermatozoa  Shape represents the tubular lumen: parallel sides,
2. Bacteria rounded ends, inclusions/additional elements from
3. Yeasts the filtrate
4. Parasites  Detected under LPO, identification on HPO
5. Mucus  Scan edges of glass coverslip
 Report number per lpf
SPERMATOZOA  Many pathologic and non-pathologic causes
 Normally seen in urine after sexual intercourse, 
nocturnal emissions or masturbation
 Significance: male infertility or retrograde ejaculation  Why is low light essential in examining for casts?
(semen enter the bladder instead of penis during Casts have low refractive index.
orgasm)
COMPOSITION AND FORMATION OF CASTS
 Tamm-Horsfall protein (THP)/Uromodulin – primary
BACTERIA
 Urine is usually sterile, contaminated on the way out;
composition; secreted by RTE of DCT and collecting
contaminants multiply fast duct
 Other components: albumin, immunoglobulin
 Indicative of lower or upper UTI when seen with WBCs
 Excretion of the THP is consistent; increased excretion
in fresh specimens
 Most common: Escherichia coli
caused by: increased stress and exercise
 THP is not detected by reagent strips
 Nitrate helps to confirm the presence of rods, but not
cocci
 Why are protein reagent pads usually positive when
casts are detected?
YEASTS The primary protein is THP but albumin and immunoglobulin can
 Single, refractile, budding structures also be incorporated as matrix. Regent strip are (+) for albumin
 Mycelial forms may be present which is why the reagent pad also turn (+).
 Diabetic urine: increased glucose and acid which is
ideal for yeast growth  Contributing factors in the formation of casts/gelling
 Seen in immunocompromised, vaginal moniliasis of THP
1. Urine stasis or obstruction of the nephron
 Presence of yeast cells without WBCs signifies? 2. pH; high levels of sodium and calcium
It may not be a true yeast infection but just contamination. 3. electrolyte and protein constituents
 Formation: Forms at the junction of ascending loop of
PARASITES Henle and DCT
 Usually contaminants from vaginal or fecal matter: – Aggregation of THP into individual protein fibrils
 Schistosoma haematobium: inhabits vein in urinary attached to RTE cells
bladder wall – Interweaving of protein fibrils to form loose
 Trichomonas vaginalis: most common; pear-shaped network which traps elements
flagellate; swims across field very rapidly – More interweaving to form solid matrix
 Enterobius vermicularis: D-shaped ova – Attachment of elements to matrix
– Detachment of fibrils from RTE cells
MUCUS – Excretion of cast
 Protein from RTE, glands, squamous cells  Cylindroids – casts with tapered ends; same
 Threadlike, low refractive index significance as casts
 Confused with hyaline casts  Cylinddruria/Cylindriduria – presence of urinary
 More frequent in females, no clinical significance cylindroids/casts

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TYPES OF CASTS  Hemoglobin degraded to methemoglobin =
1. Hyaline Casts granular dirty brown casts
2. RBC Casts
3. WBC Casts WBC CASTS
4. Mixed Cellular Casts  Mostly composed of neutrophils and lobed nucleus
5. Bacterial Casts and granules are seen
6. RTE/Epithelial Cell Casts  Staining helps differentiate from RTE cells
7. Fatty Casts  May be tightly packed; look for cast matrix to
8. Granular Casts distinguish from WBC clump
9. Waxy Casts  Clinical significance:
10. Broad Casts  Seen with infection and/or inflammation of the
tubules
HYALINE CASTS  Pyelonephritis, acute interstitial nephritis (AIN),
 Most frequently seen cast in urine; composed entirely glomerulonephritis (may be seen in conjunction
of THP with WBCs)
 Colorless when unstained and often overlooked
because they have the same refractive index as urine  Will there be WBC casts in urinary tract infections?
(low); when stained using SHM, they appear pink
 Using the phase-contrast microscopy, the visualization MIXED CELLULAR CASTS
increases  Consists of different cell types
 Have normal parallel sides on convoluted, wrinkled,
cylindroid, and occasional adhering cell or granule  What other casts may be seen with mixed cellular
casts?
 0-2 lpf is normal
 Clinical significance:
BACTERIAL CASTS
 Physiologic: stress, exercise, fever, heat exposure,
 May be pure bacteria or mixed with WBCs
dehydration
 Identification can be difficult because it resembles
 Pathologic: glomerulonephritis, pyelonephritis,
granular casts so consider the presence of free WBC
chronic renal disease, congestive heart failure
and bacteria; confirm with Gram staining
 Clinical significance:
RBC CASTS
 Seen in pyelonephritis
 Appear as orange-red color under LPO; more fragile
 Mixed cellular casts in glomerulonephritis: RBCs
than other cast
and WBCs
 Embedded and adhering cells but may also be
fragmented  Will there be bacterial casts in urinary tract infections?
 Look for cast matrix (confused with RBC clump )
 Clinical significance:
 Look for predominant type of cell: Neutrophils?
 Presence of RBC casts in urine indicates bleeding
Eosinophils? Lymphocytes?
within the nephron, casts are more specific than
free RBCs in urine RTE/EPITHELIAL CELL CASTS
 Glomerular damage, nephron capillary damage,
 Formed due to urine stasis and RTE desquamation
tubular necrosis  Fibrils forming cast pull cells from damaged tubules
 Glomerular damage: seen in conjunction with
 Formed in DCT = small, round, differentiate from WBC
proteinuria and dysmorphic RBCs  Majority of cells are on the cast matrix
 Tubular necrosis: seen in conjunction w/fatty casts
 Look for matrix to distinguish fragments
 Physiologic: strenuous activities
 Clinical significance:
 Tubular damage, heavy metals, viral infections,
 If there is bleeding within the nephron, RBC casts are
more specific than free RBCs in urine, why? drug toxicity, graft rejection, pyelonephritis
 Hepatitis
 Glomerulonephritis: cells may appear bilirubin stain
 Cells begin to disintegrate with more stasis of urine
flow
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FATTY CASTS  May be 2-6x wider than regular casts and can be
 Seen with free fat droplets and oval fat bodies cellular, waxy, or granular in composition
 Highly refractile elements, which have incorporated  All types of casts: most common are granular and
either fat droplet or OFBs that may attach to matrix waxy
 Confirmation is via polarized microscopy and lipid  Formation: due to extreme urine stasis
stains aids in identification  Destruction and widening of the DCTs
 Clinical significance:  Formation in the upper collecting duct
 Seen in: nephrotic syndrome, diabetes, crush  Clinical significance:
trauma, toxic tubular necrosis  Indicates grave prognosis
 Also seen in tubular necrosis caused by viral
GRANULAR CASTS hepatitis
 Second most common type of cast
 Can result either from the breakdown of cellular cast Artifacts Resembling Casts
or the inclusion of aggregates of plasma protein or  Material fibers, meat and vegetable fibers, and hair
immunoglobulin; may be coarse or fine  Casts do not polarize except fatty casts
 Granule origin:  Many fibers polarize
 Lysosomes: excreted in normal metabolism
 Disintegration of cellular casts and free cells ARTIFACT INTERFERENCE
 Clinical significance:  Large pollen grain
 Physiologic: stress and strenuous activities No usual sediment elements in view
 Glomerulonephritis, pyelonephritis, other  Grain is in a different liquid plane than the urine
conditions with urine stasis (fine) constituents due to its larger size

WAXY CASTS Urinary Crystals

 Brittle, highly refractile; often fragmented with jagged  Formed by precipitation of urine solutes: inorganic
ends and notches; well visualized with stain salts, organic compounds, and medications/iatrogenic
 Clinical significance: compounds
 Degenerated hyaline and granular casts due to urine  Most are not clinically significant but are reported
stasis indicating chronic renal failure  Normal crystals must be differentiated from the few
 Other associated conditions: Amyloidosis, advanced abnormal crystals indicating liver disease, inborn
nephritis errors of metabolism, and damage to tubules
 Report: rare, few, moderate, many
 Can waxy casts be seen without any other type of  Factors affecting precipitation/solubility of crystals:
casts? Why? temperature, solute concentration, pH
 General Identification:
 When cellular casts breakdown, they become coarse  Most have characteristic shapes and colors
granular casts. If there is urine stasis, it will become  Most valuable id is urine pH
fine granular casts, and then turns into waxy casts  Classification: normal acid, normal alkaline
when final degradation occurs.  All abnormal crystals are found in acid urine
 Polarized microscopy characteristics are valuable in
BROAD CASTS identification
 Known as “renal failure casts”

URINARY CRYSTALS
CRYSTAL APPEARANCE/DESCRIPTION CLINICAL SIGNIFICANCE OTHER NOTES
NORMAL CYRTSAL IN ACIDIC URINE: MOST ENCOUNTERED CRYSTALS
 Yellow-brown granules
 Urine sediment has pink color due to
 May be seen in acidic and neutral
Amorphous uroerythrin attaching in surface of  Non-pathologic
urine
urates granules  Common in refrigerated specimens
 Dissolved by alkali and heat
 Often in clumps; may resemble casts
 pH: >5.5

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 Usually non-pathologic
 Clumps: renal calculi
 Monohydrate form: ethylene
Calcium  Dihydrate (most common): envelope,  May be seen in neutral and alkaline
glycol/anti-freeze poisoning
oxalate two pyramid-shaped urine
 Sources of high oxalic acid: oranges,
(CaOx)  Monohydrate: oval dumbbell-shaped  Dissolves in dilute hydrochloric acid
tomatoes, spinach, rhubarb, garlic
and asparagus, and high doses of
ascorbic acid
 Yellow-brown, colorless, red-brown  Usually non-pathologic
 Rhombic, whetstones, wedges,  Associated with gout in large  Dissolved by alkali
rosettes, square, lemon-shaped numbers  Best identified using polarized light
Uric acid
 May resemble cysteine crystal but  Lesch-Nyhan Syndrome = multicolored
always polarize  Increased amounts in fresh urine:  Seen in pH <5.5
 High purine, nucleic acid chemotherapy (leukemia patients)
NORMAL CRYSTAL IN ACIDIC URINE: LESS ENCOUNDERED CRYSTALS
 Colorless, yellowish needles, slender
 Little clinical significance
Sodium prisms, occurring in clusters  Seen together with acid urates and
 Seen in synovial fluid due to gout but
urates  Arranged in a can or “peacock tail amorphous urates
may also be seen in urine
crystals”
 Larger granules and may have
 Seen together with sodium urates in
Acid urates spicules which are similar to  Little clinical significance
less acidic urine
ammonium biurate
 Rarely seen and only appears due to
 Yellow-brown, colorless elongated eating food with large amounts of  May be seen in neutral alkaline urine
Hippuric acid
prisms, plates benzoic acid  Dissolved by water and ether
 No clinical significance
 Long thin, colorless needles, prisms
Calcium identical to calcium phosphate  Rarely seen
 Dissolved in acetic acid
sulfates  Cigarette butt-like, star-like  No clinical significance
appearance
NORMAL CRYSTALS IN ALKALINE URINE
 May appear similar to amorphous
urates
Amorphous  To differentiate:  Non-pathologic  Seen in neutral urine
phosphates – Seen in alkaline pH and heavy  Common in refrigerated specimens  Dissolved by acetic acid but not heat
white precipitate after
refrigeration
Struvite/
 Usually non-pathologic
Triple  Colorless, prism, “coffin-lid-shaped”
 Associated with: urine stasis in
phosphate/  Has feathery appearance when  Dissolved by dilute acetic acid
kidney and bladder, chronic UTI,
Magnesium disintergrated  Also seen in neutral/acidic urine
urea-splitting bacteria, 10-20% of
ammonium  Polarize
renal calculi
phosphate
 Yellow-brown, golden, spicule-  Non-pathologic  Dissolved by acetic acid/heat (-60°C)
Ammonium
covered sphers, “thorny apples”  Associated with the presence of urea-  Converts to uric acid crystals when
biurate
 Only urates in alkaline urine splitting bacteria, stale urine added with glacial acetic
 Also seen in neutral urine
 Small, colorless, dumbbell, sphere-
Calcium  Usually non-pathologic  Can be confused with amorphous
shaped
carbonate  Associated with calculi formation phosphates
 Larger than amorphous phosphates
 Dissolved by acetic acid (gas)
Apatite/  Flat rectangles and thin prisms in
 Usually non-pathologic
Calcium rosettes, stars, “stellar phosphates”  Dissolved by dilute acetic acid
 Associated with calculi formation
phosphate  Confused with sulfonamide
ABNORMAL CRYSTALS IN ACIDIC URINE
 Hexagonal, refractile, thin, thick
plates
 May appear singly, top of each other,  Cystinuria (tendency to form renal  Thick cysteine can only polarize
Cysteine
cluster, but frequently has a layered calculi = staghorn calculi = stasis)  Confused with uric acid
or laminated appearance
 Confirm: cyanide nitroprusside

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 Rectangular, flat, transparent plates
with characteristics notched corners,  Associated with conditions producing  Rarely seen unless specimens have
“staircase pattern” lipiduria (nephrotic syndrome) been refrigerated
Cholesterol
 Highly birefringent  Seen in conjunction with fatty casts  Dissolved by chloroform, ether and
 Under polarized light may exhibit and OFBs hot alcohol
variety of colors
ABNORMAL CRYSTALS IN ACIDIC URINE: LIVER DISEASE CRYSTALS
 Yellow, clumped needles, granules
 Hepatic disorder  Correlate with bilirubin chemical test
 In viral hepatitis causing tubular  Dissolved by acetic acid, hydrochloric
Bilirubin
damage, may be found incorporated acid, sodium hydroxide, ether and
into the cast matrix chloroform
Hepatic disorders
 Yellow-brown spheres w/ concentric More common than tyrosine  Dissolved by heat and alkali, alcohol
Leucine
circles and radial striations Never seen without tyrosine  Not dissolved by hydrochloric acid
Amino acid metabolism disorder
Hepatic disorders
Commonly seen together with leucine
 Fine yellow needles in clumps or  Dissolved by heat or alkali
Tyrosine crystals w/ (+) chem test in bilirubin
rosettes  Cause sour/rancid odor
 Inherited amino acid metabolism
disorder
ABNORMAL CRYSTALS IN ACIDIC URINE: IATROGENIC CRYSTALS
Radiographic  Differentiated from cholesterol  Resembles cholesterol crystals
 Extremely high SG: >1.040
contrast crystal by UA results and patient  Seen after procedures using
 Dissolved by 10% sodium hydroxide
media history radiographic dye
 Variety of shapes: needles, rhombic,
 Common cause: high doses of
whetstone, rosette  Green color
sulfonamides with inadequate
Sulfonamides  Colorless, yellow-brown  Dissolved by acetone
hydration
 Confused with calcium phosphate  Patient history to help ID
 May suggest tubular damage
– Confirm: Lignin test
 Massive doses of penicillin
 Colorless needles that form bundles
Ampicillin compounds with inadequate  Patient history to help ID
after refrigeration
hydration

CHAPTER 7: RENAL DISEASES

INTRODUCTION 3. Glomerular basement-membrane thickening
 Viscous disorders throughout the body can affect renal 4. Hyalinization of the glomeruli
function and produce abnormalities in the urinalysis.  Non-immunologic
 Chemicals and toxins, deposition of amyloid
 Why are the kidneys frequently affected by material and acute phase reactants, electrical
complications from these various disorders? charge interference, membrane thickening
 Glomerular injury is due to toxic substances produced
MAJOR CLASSIFICATION OF RENAL DISEASES by complement protein, WBCs, platelets, etc. at the
site of antibody deposition
Glomerular  Affects the glomerular filtration barrier causing
 Majority are of immune origin
>2g/dL of protein in urine
 Immune complexes from immunologic disorders
 Glomerulonephritis
throughout the body trapped in the glomeruli – General term for sterile, inflammatory process
 Increased serum IgA; antibodies react directly with
affecting the glomerulus, associated with
glomeruli tissue hematuria, proteinuria and cylindriduria and some
 Immune system mediators (complement, etc);
or all other clinical features (azotemia, edema,
morphologic changes due to glomerular disorders: hypertension)
1. Leukocyte-membrane infiltration
– Causes blood, protein, and casts in urine
2. Cellular proliferation

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 Types of glomerulonephritis progress through various  Inherited – lack of gene/s for reabsorption
disorders  Metabolic – override of maximal re-absorptive
– Acute glomerulonephritis to chronic capacity
glomerulonephritis (CGN) to nephrotic syndrome
(NS) to renal failure Interstitial
 a.k.a Tubulointerstitial Disease
Tubular  Primarily involves infections and inflammatory
 Caused by actual damage to the tubules conditions
 Due to inherited and/or metabolic disorders  Pyelitis vs pyelonephritis

RENAL DISORDERS
DISORDER ETIOLOGY CLINICAL COURSE LABORATORY RESULTS OTHER NOTES
GLOMERULAR NEPHRITIS
 Sudden onset of fever,  Macroscopic hematuria
 Deposition of immune complexes  Group A
Acute (Post- edema around eyes,  Dysmorphic RBCs
on the glomerular membranes streptococcal
Streptococcal) fatigue hypertension,  Hyaline, granular and RBC casts
 More common in children and infections from
Glomerulo- oliguria and  Proteinuria
young adults following organisms with M
nephritis hematuria  Early: increased BUN
respiratory infections with protein in the cell
(AGN)  Permanent renal  Serological tests
Streptococcus pyogenes wall
damage seldom occurs  (+) anti-streptolysin O (ASO)
 More serious acute
form and often has
poorer prognosis
leading to renal
failure
 Systemic immune
disorders (systemic
 Macroscopic hematuria
lupus erythro-
 Proteinuria
 Rapid onset causing matosus [SLE])
Rapidly  Deposition of immune complexes  RBC casts
glomerular damage – Macrophages
Progressive in the glomerular membranes due  Serum: increased BUN and
with more common damage
(Crescentic) to systemic immune disorders or creatinine, low GFR
progression to end- capillary walls
Glomerulo- complication of another form of  Some forms: Increased fibrin
stage renal disease – Fibrin:
nephritis glomerulonephritis degradation products,
(ESRD) permanent
cryoglobulins, deposition of IgA
damage to
immune complexes
capillary tuffs
 UA similar to AGN
progresses to more
abnormal, high-
protein, low
glomerular
filtration rate (GFR)
 Cytotoxic autoantibody: the anti-
glomerular basement membrane
 Hemoptysis and  Autoimmune
antibody is formed after a viral  Macroscopic hematuria
dyspnea followed by disorder against
Goodpasture’s respiratory infection  Proteinuria
hematuria with glomerular and
Syndrome  Attaches to the membranes of the  RBC casts
possible progression alveolar basement
glomerulus and the alveoli,  Serological tests
to CGN or ESRD membranes
activating the complement,
causing capillary destruction
 Inflammation and
 Autoantibody: anti-neutrophilic  Pulmonary symptoms
granulomas in
cytoplasmic antibody (ANCA) (hemoptysis) followed
Vasculitis:  Macroscopic hematuria small blood vessels
 Attaches to the neutrophils in the by renal involvement
Wegener’s  Proteinuria of lungs and kidney
vascular walls causing damage to (hematuria,
Granulo-  RBC casts  Neutrophils initiate
the small blood vessels of the proteinuria, RBC casts,
matosis  Serological tests immune response,
kidney and lungs result to elevated BUN) and
producing
granuloma formation possible ESRD
granulomas

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 Initial appearance of
purpura (raised, red
patches on skin)
followed by blood in
Vasculitis:  Primarily affects children after a  Macroscopic hematuria  a.k.a IgA Vasculitis
sputum and stool,
Henoch- viral respiratory infection causing  Proteinuria  Follow patients for
eventually renal
Schöenlein decrease in platelets affecting  RBC casts more serious renal
involvement
Purpura vascular integrity  Stool: ___ problems
 May progress to ESRD
but most completely
recover (50%
recovery)
 Thickening of the glomerular
membrane following IgG immune  Either slow recovery
Membranous complex deposition due to or slow progression to  Microscopic hematuria
Glomerulo- systemic disorders: SLE, Sjögren NS  Proteinuria (very high)
nephritis syndrome, Hepa B, 2° syphilis,  Tendency toward  Serological tests
gold/mercury treatment and thrombosis
malignancy
 Type I: thickening
of capillary walls;
progresses to NS
 Type II: glomerular
Membrano-  Cellular proliferation affecting the  Hematuria basement
 Slow progression to
proliferative glomerular basement membrane  Proteinuria membrane
either chronic GN (II)
Glomerulo- and/or peripheral capillaries;  Serology: decreased thickening;
or NS (I)
nephritis possibly due to immune reactions complement levels progresses to CGN
 Autoimmune
disorders,
infections,
malignancies
 Gradually worsening
 Hematuria
symptoms: fatigue,
Chronic  Marked by decreased in renal  Proteinuria
anemia, hypertension,
Glomerulo- function resulting from  Glycosuria
edema, oliguria
nephritis glomerular damage due to other  Cellular, granular, waxy and
 Noticeable decrease in
(CGN) renal disorders broad casts (telescopic urine)
renal function
 Serum: increased BUN
progressing to ESRD
 Common in children and young
 Recurrent macroscopic
IgA adults
hematuria following  Early: macroscopic/ microscopic
Nephropathy  Deposition of IgA on the
exercise with hematuria  Most common
(Berger glomerular membrane due to
spontaneous recovery  Serology: increased IgA levels cause of GN
Disease) increased serum IgA levels; it
 Slow progression to  Late: see CGN/ESRD
may also be caused by mucosal
CGN or ESRD
infection
 Glomerular
membrane damage
 Edema and
and changes un
hypertension  Microscopic hematuria
 Disruption of the shield of podocyte electrical
 Acute onset following  >3.5g/day proteinuria
negativity and damage to the charges
Nephrotic systemic shock  RTE cells, OFB, fat droplets,
tightly fitting podocyte barrier  Protein passes
Syndrome  Gradual progression fatty and waxy casts
resulting in massive loss of through
from other glomerular  Serum: decreased albumin,
protein and lipids membrane; serum
disorders then to cholesterol and triglycerides
albumin depleted,
renal failure
causing increased
lipid production
 Disruption of the podocytes
Minimal  Edema  Transient hematuria
occurring primarily in children (2-  Not very much
Change  Frequent complete  Heavy proteinuria
6 y/o) associated with
Disease (Lipid remission after  Fat droplets
 Unknown etiology but associated cellular change in
Nephrosis/ Nil corticosteroid  Serum: decreased albumin,
with allergic reactions, the glomerulus
Lesion) treatment cholesterol and triglycerides
immunization, HLA-B12
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 Disruption of podocytes in certain
Focal  Course may resemble  Microscopic hematuria
areas of glomeruli associated
Segmental either nephrotic  Heavy proteinuria
with AIDS (HIV), as well as heroin
Glomerulo- syndrome or minimal  Other tests: HIV testing, drugs
and analgesic abuse
nephritis change disease of abuse (heroin, analegesic)
 Usual immune deposits: IgM, C3
 Causes macroscopic
hematuria during
respiratory infections  Inherited sex-
 Uncommon, mostly affects males  Early: macroscopic or
in males <6 y/o linked and
Alport  Genetic disorder of collagen microscopic hematuria
 Abnormal hearing or autosomal disorder
Syndrome production causing laminated and  Other test DNA tests
vision may also be affecting basement
thinning glomerular membrane  Late: see NS
present membrane
 Slow progression to
NS and ESRD
 a.k.a. Kimmelstiel-
Wilson disease
 Increased
proliferation of
 Complication of DM
 Most common cause of mesangial cells
 Glomerular basement membrane
ESRD  Early: microalbuminuria  Increased
Diabetic thickening
 Controlled diet and BP  Serum: increased FBS deposition of
Nephropathy  Deposition associated with
decrease progression  Late: see CGN cellular and
glycosylated proteins from poorly
to ESRD acellular material
controlled diet
within matrix of
Bowman’s capsule
and around
capillary tufts
TUBULAR DISORDERS
 Damage to the renal tubular cells
caused by:
1. Ischemia – severe decrease in
blood flow
– Shock (cardiac failure,  Microscopic hematuria
 Various courses
toxigenic sepsis,  Mild proteinuria
 Acute onset of renal
Acute Tubular electrocution, massive  RTE cells and casts
dysfunction usually
Necrosis hemorrhage, anaphylaxis)  Hyaline, granular, waxy and
resolved when
(ATN) trauma/crush injuries broad casts
underlying cause is
2. Nephrotoxic agents –  Others: Hgb/Hct, cardiac
corrected
cyclosporine, aminoglycosides, enzymes
amphotericin B, radiographic
dye, organic solvents, heavy
metals, toxic mushrooms,
hemoglobin and myoglobin
 Failure of tubular reabsorption in
PCT
 Inherited in association with  Glycosuria
 Generalized defect in
cystinosis and Hartnup disease  Cysteine crystals sometimes
Fanconi renal dysfunction
 Complication of multiple myeloma  Others: serum/urine
Syndrome requiring supportive
or renal transplant electrolytes, amino acid
therapy
 Acquired through exposure to chromatography
toxic agents: heavy metals,
outdated tetracycline
 Inherited defect in the production  Continual monitoring
Uromodulin-
of normal uromodulin by the renal of renal function for
Associated  Early: RTE cells
tubules and increased uric acid progression to renal
Kidney  Serum: high uric acid levels
causing gout as early as teenage failure and possible
Disease
years kidney transplantation
 Inherited defect of tubular  Pale yellow urine  Two types:
Nephrogenic  Requires supportive
response to ADH  Low SG 1. Nephrogenic –
Diabetes monitoring therapy to
 Complication of PCKD  Polyuria failure of tubules
Insipidus prevent dehydration
 Acquired from medications:  Others: ADH testing to respond to
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lithium, amphotericin B ADH, inherted
sex-linked
recessive or
lithium and
amphotericin B
exposure,
polycystic
kidneys and
sickle cell
anemia
2. Neurogenic –
failure to
produce ADH
Renal  Inherited autosomal recessive
 Glucosuria
Glycosuria/ trait causing failure to reabsorb  Benign disorder 
 Normal blood glucose
Glucosuria glucose
INTERSTITIAL DISORDERS
 WBCs and bacteria
 Ascending bacterial infection of  Acute onset of urinary  Untreated:
 Microscopic hematuria
the bladder frequency and burning progresses to
Cystitis  Mild proteinuria
 More common in women and sensation resolved by upper urinary tract
 Increased pH
children antibiotics  No cast
 Positive urine culture
 Ascending
movement of
bacteria
– Conditions
 Acute onset of urinary  WBCs and bacteria
 Infection of the renal tubules and affecting
Acute Pyelo- frequency and burning  WBC and bacterial casts
interstitium due to interference of emptying of
nephritis sensation, and lower  Microscopic hematuria
urine flow to the bladder, or bladder
(APN) back pain resolved by  Proteinuria
untreated cystitis – Calculi,
antibiotics  Positive urine culture
pregnancy,
reflux of urine
from bladder to
ureters
 WBCs and bacteria
 WBC and bacterial casts
 Frequently diagnosed
 Granular, waxy and broad casts  Congenital
 Recurrent infection of the renal in children; requires
 Hematuria structural defects
Chronic Pyelo- tubules and interstitium due to correction of
 Proteinuria causing reflux are
nephritis structural abnormalities affecting underlying defect
 Positive urine culture most common
the flow of urine  Possible progression
 Serum: increased BUN and cause
to renal failure
creatinine, decreased GFR and
concentration
 Common initial  Medication allergy
symptoms: fever and to penicillin,
skin rash  WBCs and WBC casts methicillin,
 Acute onset of renal eosinophils ampicillin,
Acute  Allergic inflammation of the renal dysfunction: oliguria,  Hematuria cephalosporin,
Interstitial interstitium followed by edema, decreased GFR  Proteinuria NSAIDs, thiazide
Nephritis inflammation of renal tubules due and concentration  Positive urine culture diuretics;
(AIN) to certain medication  Resolves following  Serum: increased BUN and discontinue; use
discontinuation of creatinine, decreased GFR and steroids
medication and concentration  No bacteria
treatment with  Hansel stain for
corticosteroids eosinophils

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OTHER DISORDERS  Marked by: oliguria, increased BUN and creatinine,
renal failure, low sodium urine, no proteinuria and
Renal Failure abnormal sediments

CHRONIC RENAL FAILURE Renal Lithiasis

 Progression from original disorders to ESRD  Renal calculi (kidney stones) in the calyces and pelvis
 Progression marked by of the kidney, ureters and bladders, which are
 Decreased GFR (<25 mL/min) staghorn, round and smooth, barely seen
 Steadily rising serum, BUN and creatinine levels  Conditions of formation are similar to the formation of
 Azotemia urinary crystals
 Electrolyte imbalance  pH concentration, stasis
 Isosthenuria  no exact cause of formation known
 Proteinuria  increased in the summer, dehydration
 Renal glycosuria  Passing of small calculi subjects patients to severe
 Abundance of granular, waxy, broad cast (telescope pain from the lower back of the legs
urine sediment)  Larger stones are not passed and only detected when
 Stages: diminished renal failure  renal insufficiency patient develops symptoms of urinary obstruction.
 renal failure  ESRD May be removed using:
1. Surgery
ACUTE RENAL FAILURE 2. Lithotripsy – high energy shock waves break up
 Sudden onset, often reversible stones
 Primary causes:  Analysis of composition:
 Pre-renal: decreased blood flow/pressure/cardiac 1. Chemical
output, hemorrhage, burns, surgery, septicemia 2. X-ray crystallography
 Renal: acute disease (AGN, ATN, APN, AIN)
 Post-renal: renal calculi, tumors, crystallization of
CONSTITUENTS OF RENAL CALCULI
ingested substances 1. Calcium oxalate – major constituent (75%); very hard,
 General Characteristics/Findings: decreased GFR,
dark in color, rough; associated with metabolic
oliguria, edema, azotemia calcium/phosphate disorders and occasionally diet
 Urinalysis related to cause:
2. Magnesium ammonium phosphate (struvite) –
 RTE cells: decreased blood flow
frequently accompanied by UTI caused by urea-
 RBCs: glomerular damage
splitting bacteria
 WBCs and casts: infection/inflammation
3. Uric acid – yellow to brownish red, moderately hard;
 Urothelial cells: possible bladder tumor
associated with increased intake of food with high
purine
Hepato-renal Failure 4. Cysteine – yellow-brown, greasy, like old soap
 Usually occurs with fulminant hepatitis or advanced
5. Phosphate – pale, friable stone
liver cirrhosis with ascites fluid build-up

CHAPTER 8: RELATED METABOLIC DISORDERS

INTRODUCTION Classification of Metabolic Disorders According
 Either abnormal metabolites or excess metabolites to Functional Defect
from various metabolic disorders appear in the blood 1. Renal
or in the urine. a. Hartnup disease
 Renal disorders b. Cystinuria
 Overflow disorders 2. Overflow: Non-hereditary
a. Tyrosinemia in infants and of acquired liver disease
b. Melanuria

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c. Indicanuria group that branches from the main aliphatic carbon
d. 5-hydroxyindoleacetic acid chain; significant laboratory finding: ketonuria in
e. Porphyria (some) newborns
f. Carbohydrate disorders (few) c. Tryptophan Disorders
3. Overflow: Hereditary d. Cystine Disorders – noticeable sulfur odor may be
a. Phenylketonuria present in urine
b. Tyrosinemia/tyrosyluria 2. Porphyrin Disorders
c. Alkpatonuria 3. Mucopolysaccharide Disorders
d. Maple syrup urine disease 4. Purine Disorders
e. Organic acidemias 5. Carbohydrate Disorders
f. Idicanuria (Hartnup’s disease)
g. Cystinosis and homocystinuria
h. Porphyria (some)
i. Mucopolysaccharidoses
j. Lesch-Nyhan disease
k. Carbohydrate disorders (most)

Newborn Screening Tests

 Uses blood collected from infant heel puncture
 Analyzed using tandem mass spectrophotometry
 Urine tests may be used for follow-up

Biochemical Classification of Metabolites

1. Amino Acid Disorders
a. Phenylalanine-Tyrosine Disorders
b. Branched-Chain Amino Acid Disorders – differ from
other amino acids due to the presence of methyl

AMINO ACID DISORDERS

DISORDER ETIOLOGY CLINICAL MANIFESTATIONS LABORATORY TESTS TREATMENT/NOTES
PHENYLALANINE-TYROSINE DISORDERS
1. Newborn screening:  Elimination of
 Autosomal recessive trait: Undetected cases, causing phenylalanine in
detected 4h after birth if
failure to inherit patients to continually eat the diet; dietary
newborn have adequate
phenylalanine hydrolase phenylalanine-rich food causes: restriction may be
ingestion of milk; girls
which converts phenylalanine  Severe mental retardation eased as child
have higher false (–)
Phenyl- to tyrosine causing an  Peculiar mousy/musty odor of matures
results
ketonuria alternate degradative urine
2. Ferric chloride: nonspecific;
pathway which produces an  Tyrosine deficiency leading to
increased level of light skin and hair
detects PPA; color reaction:  Most well-known
permanent blue-green amino aciduria
phenylketones (primary PPA;  Microencephaly: offspring of
also, PLA, PAA, PAG) women with PKU
3. Guthrie test  1 in every 10,000-
4. HPLC (reference method) 20,000 births
 Excess tyrosine or  Rancid odor of urine
degradation products (p-  Hereditary: fatal liver and
1. Newborn screening
hydroxyphenylpyruvic acid renal tubular disease  Dietary restriction
2. Nitroso-naphthol test:
and p-hydroxyphenyllactic) a. I: generalized renal (inherited: no/low
mercuri in concentrated
1. Hereditary: failure to tubular disorder and phenylalanine and
nirtic acid; color reaction:
Tyrosylemia/ inherit genes for enzymes progressive liver failure tyrosine
orange-red precipitate
Tyrosinemia/ a. Ia: fumaryl- soon after birth  Transitory: returns
3. Millon’s test
Tyrosyluria acetoacetate hydrolase b. II: corneal erosion and to normal within
4. Ferric chloride: transient
Ib: maleylacetoacetate lesions on the palms weeks or months
green
isomer fingers and soled of the  Acquired: resolution
5. Modified Guthrie test
b. II: tyrosine feet of liver disease
6. Chromatography: confirm
aminotransferase c. III: mental retardation if
c. III: p-hydroxyphenyl- without dietary
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pyruvate dioxygenase restrictions of
Transitory: premature
2. phenylalanine and
infants (underdeveloped tyrosine
liver); most common cause  Tyrosine and leucine crystals
3. Tyrosyluria of acquired are rarely seen in both
liver disease: more transitory and acquired types
serious
 Second metabolic pathway
for tyrosine: produces  Melanin responsible for dark
melanin, thyroxine, color of hair, skin and eyes
1. Thormahlein sodium
epinephrine, poteins,  Albinism: deficient production
nitroprusside test
tyrosine sulfate  Removal of tumor
Melanuria 2. Ehrlich’s: red color
 Tumors secrete 5,6-  Chemotherapy
3. Ferric chloride: gray/black
 Proliferation of melanocytes dihydroxyindole which
precipitate
which produces a malignant oxidizes to melanogen and
melanoma then to melanin, producing
dark urine
 Failure to inherit  Not clinical in early childhood
homogentisic acid oxidase except odor 1. Newborn screening
resulting accumulation of brown/black/reddish brown 2. Clinitest: yellow  Dietary restriction:
homogentisic acid in the stained diapers precipitate no/low
blood, tissues and urine  Deposition of brown pigment 3. Alkali test phenylalanine and
Alkpatonuria
 Alkapton: alkaline lover due in body tissues (most 4. Ferric chloride: transient tyrosine, as well as
to darkening of urine after notably, the ears) deep blue large doses of
becoming alkaline from  Cartilage deposits: arthritis 5. Peper/thim-layer ascorbic acid
standing at room  Development of liver and confirmatory/quantitation
temperature cardiac disorders
CYSTINE DISORDERS
 Approximately 65% of
people affected by (1)
 Inherited: renal tubular
produce calculi early in life
disorder causing the inability 1. Cyanide-nitroprusside
 Calculi formation is less
of reabsorbing the following
common for (2)
test: 3mL urine + 2 mL Na  False positive
Cystinuria filtered amino acids: cyanide [10mins] + 5 reactions: ketones
 Cysteine is the only amino
 Cysteine, ornithine, lysine drops Na nitroprusside  and homocysteine
acid found in calculi analysis
and arginine (+) red-purple color
 Cysteine crystals in first
 Cysteine and lysine only
morning or concentrated
specimens
 (1) Incomplete metabolism =
crystalline deposits of Routine urinalysis:
Inherited: cysteine in many areas of the  Polyuria, isosthenuria  Renal transplants
1. a. Infantile nephropathic body (BM, LN, cornea, internal  General aminoaciduria  Cysteine-dpeleting
b. Late-onset nephropathic organs)  Cysteine crystals medication
– Both has defect in the  Major defect in the renal
Cystinosis lysosomal membrane tubular reabsorption (Fanconi 1. Cyanide-nitroprusside
prevents the release of syndrome)  Cystine deposits
2. Clinitest: presence of interfere with the
cysteine to cell cytoplasm for  Photophobia, rickets, stunted
reducing substances
metabolism growth reabsorption
2. Non-nephropathic  Renal failure: rapid/gradual
especially in the PCT
 (2) May cause ocular
 NBS? Not in the book
disorders
1. Newborn screening
2. Cyanide-nitroprusside*  Dietary restriction:
 Failure to thrive 3. Silver-nitroprusside test: exclusion of foods
 Inherited: deficiency of high in methionine
 Cataracts 1mL urine + 2 drops
Homo- cystathionine-B-sythase  kwento
 Mental retardation concentrated NH4OH
cystinuria causing the accumulation of
 Thromboembolism +0.5mL %% silver
homocysteine
 Death nitrate [10mins] + 5  Fresh urine should
drops Na nitroprusside  be used
(+) red-purple color

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BRANCHED-CHAIN AMINO ACID DISORDERS
 Autosomal recessive trait:
1. Newborn screening
failure to inherit branched-
2. Microfluorometric assay
chain A-keto acid  Strong odor: maple syrup or  Dietary regulation
3. 2,4-
dehydrogenase (BCKDH) burnt sugar and careful
dinitrophenylhydrazine
Maple Syrup which are necessary for the  Failure to thrive after 1 week monitoring of
(DNPH): nonspecific *keto
Urine Disease oxidaticve decarboxylation of  Mental retardation if not urinary keto acid
(+) yellow precipitate
the transaminated leucine, detected within 11d levels can control
4. Ferric chloride: green-gray
isoleucine and valine causing  Death the disorder
5. Modified Guthrie test
their accumulation in blood
6. Chromatography: confirm
and urine
Accumulation of organic acids
formed in the latter part of
General:  2&3-metabolism
 Early sever illness often errors of converting
amino acid metabolism
with vomiting and 1. Newborn screening isoleucine, valine,
1. Isovaleric academia:
metabolic acidosis 2. Chromatography threonine,
Organic buildup of
 Hypoglycemia 3. p-nitroaniline test (for methionine to
Acidemias isovalerylglycine;
 Ketonuria methylmalinic acid) succinylcoenzyme A
deficiency of isovaleryl
coenzyme A
 Increased serum ammonia (+): emerald green  2 is immediate
Isovaleric academia: precursor of #3 in
2. Propionic acidemia
sweaty feet odor of urine the said pathway
3. Methylmalonic academia
TRYPTOPHAN DISORDERS
Increase of tryptophan  General: urine turns blue  Resolution of
1. Obermayer’s test: ferric
converted to indole causing when oxidized (blue underlying
chloride, HCl, chloroform
excess intestinal reabsorption diaper/Drummond’s disorders
(+) deep blue/violet
may be due to: syndrome)  Hartnup’s: Dietary
Indicanuria 2. Jaffe’s test
1. Intestinal obstruction  Hartnup’s: also affects the supplements
3. Jolle’s test
2. Abnormal intestinal bacteria tubular reabsorption of especially niacin
3. Malabsorption syndromes amino acids (Fanconi  Hartnup = IEM but maybe  Generalized
4. Hereditary: Hartnup disease syndrome) no NBS = *blue diaper
aminoaciduria
 Carcinoid tumors involving  Increase of 5-HIAA levels in
argentaffin/enterochromaffin urine; normally 2-8 mg/24h 1. Sjoerdsma test/nitroso- 24h sample: HCl or
cells produce excess  >25m (`160-600) mg/24h naphthol: uses nitrous boric acid
5-hyroxy- serotonin acid (not nitric acid) First
indoleacetic *Diet: banana, pineapple,  Second metabolic pathway = (+) purple to black morning/random =
Acid tomato, avocado, mushroom, 5-OH tryptophan  complex false (–)
walnuts serotonin  5-HIAA 2. Ferric chloride: (+) blue- Withhold medication
*Medications: phenothizines and  Argentaffin/entero- green for 72h
acetanilids chromaffin; platelets

OTHER DISORDERS
CLINICAL
DISORDER ETIOLOGY LABORATORY TESTS TREATMENT/NOTES
MANIFESTATIONS
1. Ehrlich reaction: for ALA  Portwine: more
and porphobilinogen prevalent for
2. Fluorescence (UV light: erythropoietic
 Inherited: rare; failure to  Acetylacetone: ALA 
550-600nm): other
inherit the gene for an  Classified as either Porpho prior to Ehrlich
porphyria
enzyme needed for the neurologic (psychiatric), a. Watson-Schwartz
– Extraction: glacial
metabolic pathway of cutaneous (photosensitivity), b. Hoesch
acetic acid and ethyl
Porphyrin porphyrins or both
Disorders/  Acquired:  Indication of porphyrinuria:
acetate  Porph: most useful
– Solvent layer: (–) acute attack, Hoesch
Porphrias erythrocytic/hepatic red or port wine urine after
faint blue (+)  Fluor: interfere subs-
malfunctions (iron deficiency, exposure to air
violet/pink organic layer removed
chronic liver disease, renal  Staining of teeth/bones
– Does not distinguish +0.5mL HCl =
disease) or exposure to toxic  Hemolytic anemia
the primary porphyrins extracted to
agents (lead, ethanol)
porphyrins but rules acid = bright orange-
out the precursor red
substances  Vampire
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 Products common in urine:
 Inherited: failure to inherit
a. Dermatan sulfate 1. Acid-albumin 30 minutes
the gene for enzyme needed
b. Keratin sulfate 2. Cetylmethylammonium
for the complete breakdown
Mucopoly- c. Heparin sulfate bromide (CTAB) turbidity
of the polysaccharide portion  Bone marrow
saccharide  Manifestation 5mins
of the compounds  transplants
Disorders/ a. Abnormal skeletal (+): thick white turbidity
accumulation in lysosomes of  Gene replacement
Mucopoly- structure Graded from 0-4
connective tissue cells + therapy
saccharidoses b. Severe mental 3. Metachromatic staining
increased excretion in urine
retardation spot test
 Most common syndromes:
c. Corneal accumulation (+) blue spot
Hurler, Hunter, Sanfilippo
d. Fatality in childhood
 Uric acid accumulation
 Development is normal
 Inherited: sex-linked throughout the body
Purine for the first 6-8mon
recessive failure to inherit  Gout and renal calculi
Disorders  First sign: increased uric
gene to produce  Severe motor defects
(Lesch-Nyhan acid crystals in pediatric
hypoxanthine guanine  Mental retardation
Disease) urine specimens
phosphoribo-syltransferase  Tendency toward self-
 Orange sand in diapers
destruction
*Mellituria: increase in urinary
 Most does not cause  Removal of the specific
Carbohydrate sugar 1. Clinitest
disturbance to body carbohydrate from the
Disorders  Inherited: more common 2. Chromatography
metabolism diet
 Acquired: multiple causes

Porphyrins  Specimen: urine, bile, feces, or blood

 Intermediate compounds in heme (protoporphyrin IX  Utilizes solubility properties and appearance in
+ iron) production body fluids:
 Formed in the bone marrow and the liver 1. Urine
 Primary porphyrins: uroporphyrin, portoporphyrin, 2. Feces: coproporphyrin and protoporphyrin; prone
coproporphyrin to false (+)
 Porphyrin precursors: A-aminolevulinic acid (ALA), 3. Bile: same as feces; more acceptable
porphobilinogen 4. Blood: FEP for lead poisoning screening
 May be blocked at several stages, causing the
accumulation of the product formed immediately Mucopolysaccharides
before the interruption  a.k.a. glycosaminoglycans: group of large compounds
 Disorders are collectively termed porphyrias located primarily in connective tissues
 Detection and identification of product to determine  consist of protein core with numerous polysaccharide
the cause: branches

CHAPTER 9: CEREBROSPINAL FLUID ANALYSIS

INTRODUCTION – Three layers of meninges:
rd
 Cerebrospinal fluid (CSF) – 3 major body fluid; 1. Dura mater – “hard mother;” outer layer; lines
clear, colorless fluid produced by the highly vascular the skull and vertebral canal
choroid plexuses in the ventricles of the brain 2. Arachnoid – “spiderweb-like;” filamentous
inner membrane
Functions of CSF 3. Pia mater – “gentle layer;” thin membrane
1. Supply nutrients to the nervous tissue lining the surfaces of the brain and spinal cord
2. Remove metabolic wastes
3. Produce a mechanical barrier to cushion the brain and  Where does the CSF flow?
spinal cord against trauma
 Choroid plexus
Formation and Physiology  Specific part of the brain that produces CSF (by
 Meninges – lining of brain and spinal cord selective filtration)
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 20 ml/h = rate of CSF production  Other methods of collection: ventricular puncture,
 CSF total volume cisternal puncture, lateral cervical puncture
– Adults: 90-150 mL  Volume of CSF removed is dependent on:
– Neonates: 10-60 mL 1. Volume available in the patient (adult vs. neonate)
 Arachnoid villae/granulation – reabsorbs CSF at 2. Opening pressure of the CSF taken when the needle
the same rate of production first enters the subarachnoid space
 Blood brain barrier  Normal opening pressure of 50-200 mmHg: up to
 Protects the brain from chemicals and other 20 mL for adults
substances circulating in the blood that can harm  Pressure >200 mmHg: 1-2 mL only
the brain tissue  Ideally, tests are done immediately, if not, it should
 Disruption of blood-brain barrier (BBB) allows WBCs, be stored at appropriate temperatures
proteins and other chemicals to enter the CSF  CSF tubes:
1. Tube 1: Chemistry/Serology (frozen)
Specimen Collection and Handling 2. Tube 2: Microbiology (room temperature)
 Most common method of collection: lumbar 3. Tube 3: Hematology/Cell Count (ref)
puncture/spinal tap 4. Tube 4: Microbiology/Serology
rd th
 Adults: between 3 and 4
th
 Neonates: between 4 and 5th  What if only one CSF tube is collected? Microbiology
– Hematology – Chemistry/Serology

APPEARANCE OF CSF SAMPLE

APPEARANCE CAUSE SIGNIFICANCE/CORRELATION
Crystal clear/colorless Normal
Hazy WBCs (>200/L) Meningitis
Turbid Microorganism
Milky Disorders affecting the BBB
Cloudy Protein and/or lipids
Production of IgG within the CNS
Oily Radiographic contrast media
Hemorrhage
Bloody RBCs
Traumatic tap (non-pathogenic)
Oxyhemoglobin (pink) Hemorrhage
Lysed cells form traumatic tap
Hemoglobin (orange)
Old hemorrhage
RBC degradation
Bilirubin (yellow)
Xanthochromic Elevated serum bilirubin level
Carotene Increased serum bilirubin
Protein Disorders affecting BBB
Rifampicin therapy Tuberculosis treatment
Melanin Meningeal metastatic melanosarcoma
Melanin Meningeal metastatic melanosarcoma
Brown Hematoma Subdural/intracerebral hemorrhage
Methemoglobin
Protein Disorders affecting BBB
Clotted
Clotting factors Introduced by traumatic tap
Protein Disorders affecting BBB
Pellicle
Clotting factors Tubercular meningitis (overnight refrigeration)

TRAUMATIC TAP VS INTRACRANIAL HEMORRHAGE

PARAMETER TRAUMATIC TAP INTRACRANIAL HEMORRHAGE
Distribution of blood Uneven 1>2>3 Even 1=2=3
Clot formation Yes/RBC clumps No
Supernatant Clear, colorless Clear to xanthochromic
Erythrophages No Yes
D-dimer No Yes

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Clot Formation without RBCs WBC Differential Count
1. Meningitis  Identifying cell types is a valuable diagnostic aid
2. Froin syndrome  Performed on stained smear and not from cells in the
3. Blocked CSF circulation counting chamber
 Specimen should be concentrated before smearing
CSF Dilution According to Appearance using the following methods:
1. Clear – undiluted 1. Cytocentrifugation
2. Slightly hazy – 1:10  Procedure:
3. Hazy – 1:20 a. 0.1mL CSF + one drop fo 30% albumin is
4. Slightly cloudy – 1:100 added to conical chamber
5. Cloudy/slightly cloudy – 1:200 b. Cells in the fluid are forced into a monolayer
6. Bloody/turbid – 1:10,000 within a 6mm diameter circle on the slide
c. Fluid is absorbed by the filter paper blotter =
HEMATOLOGY/CELL COUNT more concentrated area of cells
 Any cell count should be performed immediately  Addition of albumin: increases the cell yield and
– WBCs (especially granulocytes) and RBCs begin to decreases the cellular distortion
lyse within 1h  Positive charge-coated slide attract cells
– 40% WBCs disintegrate after 2h  Cells from both the center and periphery of the
 Always necessary as specimens containing up to 200 slide should be examined
or 400 WBCs may appear clear  Daily control slide for bacteria: 0.2mL saline + 2
 Improved Neubauer chamber: routinely used for drops of 30% albumin
performing CSF cell counts  Cytocentrifuge recovery chart
 Formula for CSF cell counts: standard Neubauer
calculation for blood cell counts WBCS COUNTED IN EXPECTED CELLS ON
# 𝑜𝑓 𝑐𝑒𝑙𝑙𝑠 𝑐𝑜𝑢𝑛𝑡𝑒𝑑 × 𝑑𝑖𝑙𝑢𝑡𝑖𝑜𝑛 𝑓𝑎𝑐𝑡𝑜𝑟 CHAMBER CYTOCENTRIFUGE SLIDE
𝐶𝑒𝑙𝑙𝑠/𝜇𝐿 =
# 𝑜𝑓 𝑠𝑞𝑢𝑎𝑟𝑒𝑠 𝑐𝑜𝑢𝑛𝑡𝑒𝑑 × 𝑣𝑜𝑙𝑢𝑚𝑒 𝑜𝑓 1 𝑠𝑞𝑢𝑎𝑟𝑒 0 0-40
WBC Count 1-5 20-100
 Routinely performed cell count for CSF specimen 6-10 60-250
 Diluted using 3% glacial acetic acid; methylene blue is 11-20 150-250
>20 >250
added to stain the nucleus
 Reference ranges:
 Adults: 0-5 RBCs/L
2. Centrifugation
 Procedure:
 Higher in children
a. Centrifugation for 5-10
 Neonates: 0-30 mononuclear cells/L
b. Supernatant is removed and saved for
additional testing
Total Cell Count
c. Slides from suspended sediment allowed to
 Dilutions are made with normal saline
dry
 On both sides of the chamber, cells are counted in the
d. Wright’s stain
four corner and central large squares
3. Sedimentation
4. Filtration – both are not routinely used but
RBC Count
produce less cell distortion
 May be calculated by performing a total cell count,
 100 cells should be counted, classified and reported in
then subtracting the WBC count
terms of percentage
 Done only in cases of traumatic tap to correct for WBC
 If cell count is low and counting 100 cells is not
count and total protein concentration
possible, report only the number of cell types seen
 1 WBC for every 700 RBCs seen
 1mg/dl total protein concentration for every 1,200
CSF Cellular Constituents
RBCs/L
 Primarily lymphocytes and monocytes
 8mg/dL total protein concentration for every 10,000
 Adults: predominance of lymphocytes to monocytes
RBCs/L
(70:30)

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 Children: predominance of monocytes to the identification of the type of microorganism
lymphocytes (reversed ratio) causing meningitis
 Pleocytosis – increased number of normal cells;  Presence of immature leukocytes, eosinophils, plasma
considered abnormal cells, macrophages, increased tissue cells and
– Pleocytosis involving neutrophils, lymphocytes or malignant cells is also considered abnormal
monocytes, the CSF differential count is valuable in

CELL TYPE CLINICAL CORRELATION MICROSCOPY/OTHER NOTES

 Normal constituent
 All stages of development may be found
 Increased in viral, tubercular, fungal and parasitic
 Moderately elevated WBC count (<25/ L) with increased
Lymphocytes meningitis
normal and reactive lymphocytes is indicative of MS or
 Also increased in asymptomatic HIV/AIDS
other degen neuro disorder
 Reactive lymphocytes with normal cells = viral infections
 Normal constituent
Monocytes  Increased in viral, tubercular, fungal and parasitic  Found mixed with lymphocytes
meningitis, and multiple sclerosis
 Seen/increased when there is presence of RBCs in the
Macrophages  May contain hemosiderin (dark blue/black, iron) granules,
spinal fluid
(more cytoplasm hematoidin (yellow, no iron) crystals, and/or phagocytosed
 Appear within 2-4h after RBCs enter the CSF
than monocytes) RBCs appearing as empty vacuoles or ghost cells
 Frequently seen following repeated taps
 Contain cytoplasmic vacuoles following cytocentrifugation
 Seen/increased in bacterial meningitis, early stages of  Granules are less prominent than in blood smears
viral, tubercular, fungal and parasitic meningitis  Cells disintegrate rapidly
Neutrophils
 Also increased following repeated lumbar punctures, CNS  May contain phagocytosed bacteria
hemorrhage, injection of medication or radiographic dye  Pyknotic nuclei = degenerating cells which may resemble
RBCs (BM contains 1%)
 Parasitic, fungal (primarily Coccidioides immitis) infections
Eosinophils
 Introduction of foreign material (medication and shunts) into the CNS
Nonpathologically Significant Cells – common after diagnostic procedures (pneumoencephalography, fluid from ventricular laps/neurosurgery)
 Seen in singularly and in clumps
Choroidal Cells  From the epithelial lining of the choroid plexus
 Usually no nucleoli while nuclei appearance is uniform
 Usually seen in clusters
Ependymal Cells  From the lining of the ventricles and the neural canal
 Usually with nucleoli; less defined cell membranes
Spindle-shaped  Seen in clusters
 From the lining of the arachnoid
Cells  Maybe seen with systemic malignancy
 Lymphoblasts, myeloblasts, and monoblasts in the CSF  Blast cells seen in the CSF have often more prominent
are frequently seen as aserious complication of acute nucleoli than blast cell in the peripheral blood
Malignant Cells of
leukemias  Lymphoma cells resemble large and small lymphocytes
Hematologic Origin
 Lymphoma cells are also seen in the CSF and indicate usually appearing in clusters
dissemination from the lymphoid tissue  Nucleoli are present and nuclei may appear cleaved
 Metastatic carcinoma cells: primarily from lung, breast,
Malignant Cells of  Usually seen in clusters
renal or gastrointestinal origin
Non-Hematologic  Observed with fusing cell walls, nuclear irregularities and
 Primary CNS tumors: astrocytomas, retinoblastomas,
Origin hyperchromatic nucleoli
medullablastomas

CHEMISTRY CSF Protein

 Reference values for CSF chemicals are not the same  The most frequently performed chemical test on CSF
as plasma values  Reference values: 15-45mg/dL; higher values in
– Filtration process is selective infants and adults >40y/o
– The BBB controls the chemical composition of CSF
 Abnormal values result from: CSF PROTEIN FRACTIONS
– Alterations in the permeability of the BBB  Generally similar to that of serum; CSF: serum ratio
– Increased production/metabolism by the neural varies per fraction
cells in response to a pathologic condition  IgM, fibrinogen and beta-lipoprotein are not present
 CSF abnormalities seldom have the same diagnostic in normal CSF
significance as plasma abnormalities 1. Albumin – major CSF protein

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2. Pre-albumin/Transthyretin – second most b. Immunofixation (IFE) – IEF/IFE for better
prevalent; also produced by the choroid plexus resolution; followed by silver staining
3. Alpha-globulins – haptoglobulin and c. Isoelectric focusing (IEF)
ceruloplasmin
4. Beta-globulins – primarily transferrin  Why is it essential to concentrate the CSF prior to
 Carbohydrate-deficient transferrin fraction performance of most techniques?
(“tau”) is seen in CSF but not in serum
5. Gamma Globulin – primarily IgG; small amounts  Purposes
a. Identification if fluid is CSF: based on the
of IgA
appearance of tau transferrin
b. Primary purpose: detect oligoclonal bands =
CLINICAL SIGNIFICANCE OF ELEVATED PROTEIN VALUES
1. Blood-brain barrier damage
inflammation within the CNS
 The bands are located in the gamma region,
a. Hemorrhage
b. Meningitis
indicating immunoglobulin production
– Together with IgG index, valuable tool for
c. Arachnoiditis
d. Guillain-Barre syndrome
the diagnosis of multiple sclerosis
– The band remains positive during remission
e. Endocrine/metabolic disorders: myxedema, diabetic
neuropathy, uremia for MS but disappears in other disorders
 Other neurologic disorders producing CSF
2. Immunoglobulin/protein production within the CNS
a. Primary CNS tumors
oligoclonal banding that may not be present in
b. Multiple sclerosis
the serum: encephalitis, neurosyphilis,
c. Guillain-Barre syndrome
Guillain-Barre syndrome, and neoplasms
 Disorders producing both CSF and serum
d. Neurosyphilis
3. Decreased normal protein clearance from the fluid
oligoclonal banding due to BBB
4. Neural tissue degeneration: polyneuritis
leakage/traumatic tap: leukemia, lymphoma
5. Others: Cushing diseases, connective tissue disease
and viral infection
 HIV infection: produces banding in both CSF

CLINICAL SIGNIFICANCE OF LOW PROTEIN VALUES and serum representing both systematic and
1. CSF leakage/trauma
neurologic involvement
2. Recent puncture
 CSF protein electrophoresis is done in conjunction
3. Rapid CSF production
with serum protein electrophoresis, why?
4. Water intoxication

4. Myelin basic protein

METHODOLOGY  Presence indicates the recent destruction of the
1. Total CSF protein
myelin sheath protecting the axons of neurons
 Turbidity – TCA and SSA
 Used to monitor the course of multiple sclerosis;
 Nephelometry – benzalkonium chloride; adapted
detected using immunoassays (e.g. RID)
for automation
 Dye-binding – CBB and Ponceau S; also used in CSF
CSF Glucose
electrophoresis  Enters CSF via the selective transport across the BBB
 Biuret
 Reference ranges: 60-70% that od the plasma
2. Differentiation of the cause of protein elevations
 CSF/serum albumin index – reference rang: <9 =  Blood for glucose testing must be done 2h prior to the
intact BBB 9-14, 15-30, >30 spinal tap, why?
 CSF/serum IgG index – reference: varied *>0.70
= IgG production within the CNS <60, >0.77  Analysis done are the same as that of serum glucose
3. Protein fractions: electrophoresis and immunophoretic  Increased values are of little value as they happen
techniques due to increases in the plasma levels or conditions
 Techniques associated with intracranial pressure; thus, primary
a. Agarose/gel electrophoresis: most frequently clinical significance is confined to finding decreased
performed values relative to plasma levels:
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– Alterations in the mechanisms of glucose transport – CSF is centrifuged at 1500g for 15min; slides and
across the BBB cultures prepared form sediment
– Increased used of glucose by the brain cells – Cytocentrifuge = highly concentrated specimens
– Determined the causative agents of meningitis: – Despite the use of concentrated specimens, there is
classic laboratory patterns that may not be found in 10% chance that G/S and cultures will be negative;
all cases but helpful when they appear therefore, blood cultures be done as agents are
often present in both CSF and blood
CSF Lactate
 Reference ranges: 10-22mg/dL Gram Stain
 Valuable aid in determining the causative agents of  Routinely performed on CSF from all suspected cases
meningitis: increased values of lactate are more meningitis: detects bacteria and fungi
consistent than decreased values of glucose;  Still the recommended method for detecting
therefore, providing a more reliable information organisms
when the initial diagnosis is difficult  One of the most difficult slides to interpret because
 Serves as a sensitive method for the evaluation of the organisms are commonly few and easily overlooked
effectiveness of antibiotic therapy  Precipitated stain or debris may also be mistaken as
 Elevated in conditions that causes tissue destruction microorganisms
within the CNS due to hypoxia  Most common bacteria:
 Used to monitor severe head injuries  Streptococcus agalactiae: neonates
 Haemophilus influenzae: 1mon <5y/o
 Xanthochromic or hemolyzed fluid causes falsely  Neisseria meningitides: 5-29y/o
elevated lactate levels, why?  Streptococcus pneumoniae: >29y/o
 Listeria monocytogenes: immunocompromised,
CSF Glutamine elderly, neonates
 Glutamine is produced from ammonia and alpha-
 Escherichia colii: most common Gram (–) rod
ketoglutarate by brain cells  Starbust pattern: Cryptococcus neoformans
 Reference value: 8-18mg/dL; >35 mg/dL causes some
disturbance of consciousness Acid-Fast Stain/Fluorescent Antibody Stains
 Indirect method for the presence of excess ammonia
 Not routinely performed unless tubercular meningitis
in the CSF: elevated levels are associated with liver is suspected
disorders that results in increased blood and CSF
ammonia (e.g. Reye syndrome) India Ink Preparation
 Frequently requested for patients with coma of
 Performed on specimens suspected for fungal
unknown origin meningitis (Cryptococcus neoformans), usually
occurring as a complication of AIDS
 Why is CSF glutamine determination preferred over
CSF/blood ammonia testing?
Wet Preparation
 Detects motile trophozoites of N. fowleri (nonmotile if
CSF Enzyme
 CK-BB, LD isoenzymes, AST
cytocentrifuged; increased WBCs, no bacteria)

MICROBIOLOGY SEROLOGY
1. Latex Agglutination Test
 Primary role: identification of the causative agent in
 More sensitive method for the detection of
meningitis
– CSF culture serves as a confirmatory rather than a
Cryptococcus neoformans than India ink
 Confirm with culture or India ink: false (+) reaction
diagnostic procedure
 Several methods are available to provide information
(most common: rheumatoid factor)
2. Lateral Flow Assay
for a preliminary diagnosis
 Rapid method for the detection of Cryptococcus
 All smears and cultures should be performed on
concentrated specimens because often only few neoformans
 Reagent stripo coated with monoclonal Abs that
organisms are present at the onset of the disease
react with the cryptococcal polysaccharide capsule
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3. Latex Agglutination and ELISA Test Kits  Limulus lysate test
 Available for the most common agents of bacterial  Diagnosis of Gram (–) bacteria
meningitis  Reagent:
 Also available: Mycobacterium tuberculosis, C.  Blood cells (ameobocytes) of horse-shoe crab
immitis (Limulus Polyphemus)
4. Bacterial Antigen Test Ameobocytes – contain copper complex than
 Available for the most common agents of bacterial gives them blue color
meningitis not very sensitive to N. meningitides  Principle: in the presence of endotoxin,
 Should be used with hematology and chemistry amoebocytes (WBC) will release lysate (protein) =
results in diagnosis of meningitis (+) clumping/clot formation
5. Test for Syphilis – RPR, VDRL, FTA-ABS *HW = chat  All materials must be sterile (tap water:
for glucose endotoxin

CHAPTER 10: SEMINAL FLUID ANALYSIS

INTRODUCTION 6. Bulbourethral (Cowper’s) gland

Indications for Seminal Fluid Analysis SPERMATOGENESIS

1. Investigation of fertility: includes screening donor for  Formation of spermatozoa in the sertoli cells (nurse
insemination, evaluation for semen/sperm banking cells) of the semniferous tubules of the testis
2. Evaluate success of vasectomy  Controlled by FSH (initiation of spermatogenesis), LH
3. Medico-legal cases and testosterone (subsequent maturation)
 Takes approximately 90d:
Anatomy and Physiology 1. Spermatocytogenesis: mitotic division (1
spermatogonium  2 primary spermatocyte)
ANATOMY 2. Meiotic phase: (2 primary  4 secondary  8
1. Scrotum and testes: seminiferous tubules, sertoli cells spermatids moves to the epididymis)
2. Epididymis 3. Spermiogenesis: development of a flagellum (8
3. Vas deferens/ductus deferens and ejaculatory duct spermatids  8 spermatozoa *vas deferense than
4. Seminal vesicles ejaculatory ducts)
5. Prostate gland

COMPOSITION OF SEMEN
PERCENTAGE COMPOSITION NOTES
5 Spermatozoa Majority contained in the first part of the ejaculate
Slightly alkaline fluid serving as the transport medium for the sperm
60-70 Seminal fluid Has high concentration of fructose (energy) and Flavin (gray color)
Also contains citric acid and potassium
20-30 Prostate fluid Milky, acidic fluid containing high concentration of ACP, citric acid, zin, choline, spermine
5 Bulbourethral fluid Thick, alkaline mucus that helps neutralize vaginal acidity
*Mixing of all fractions is important for the production of normal semen

Specimen Collection and Handling – Sperm count, pH and liquefaction

 All semen specimens are potential reservoir for HIV
 Why is proper collection of complete specimen and hepatitis viruses
essential? Accurate evaluation, variety of composition.  Standard precautions must be observed at all times
during analysis
 Patients should receive detailed instructions for
 Sterile materials and techniques should be used for:
specimen collection semen culture, bioassay, IUI, IVF
 If a part of the first portion of the ejaculate is missing
the following will be affected:
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COLLECTION METHODS 2. Fertility testing: two-three samples (not less than 7d
1. Masturbation – preferred or more than 3wk apart) must be analyzed
2. Condom – no lubricants or polyurethane; no 3. Warm, sterile glass or plastic containers
spermicides 4. As much as possible, specimen must be collected in a
3. Coitus interruptus – not reliable; first portion and room provided by the laboratory. If this is not
vaginal pH appropriate, specimen must be kept at room
4. Vaginal vault aspiration – for ability to penetrate temperature* and delivered to the laboratory within
cervix an hour.
5. Aside for vital information (name, birthday, time
SPECIMEN REQUIREMENTS collected and received), personnel should record the
1. Abstinence: 2-7d (prolonged: higher volumes, period of sexual abstinence and the completeness of
decreased motility) the sample
6. Specimens awaiting analyses should be kept at 37°C.

MACROSCOPIC ANALYSIS

APPEARANCE OF SEMEN
APPEARANCE CAUSE/SIGNIFICANCE/CORRELATION
Translucent, gray-white Normal
Almost clear Low sperm concentration
Presence of WBCs (indicates infection within the reproductive tract
*If needed, semen culture must be done prior to semenalysis
Increased white turbidity
*Must be differentiated from spermatids during microscopy
*LE reagent strip may be used to screen for WBCs
Various red coloration Presence of RBCs
Urine contamination (toxic to sperm = motility affected)
Yellow Prolonged abstinence
medications

Odor  Jelly-like granules may be present in liquefied semen

 Musty/bleach-like (chlorox-like) and hve no clinical significance
 Mucus strands, if present, may interfere with analysis
Liquefaction
 Fresh semen specimen is clotted and liquefies within Volume
30-60min after collection  Normal volume: 2-5mL
 Failure to coagulate: may indicate congenital bilateral  Measured by pouring semen into a clean graduated
abstinence of the vas deferens and seminal vesicles cylinder with 0-1mL increments
 Failure of liquefaction within an hour may indicate:  Increased volume: prolonged abstinence
 Incomplete collection  Causes of low volumes:
 Deficiency of prostatic enzymes  Incomplete specimen collection
 If semen has not liquefied after 2h, any of the  Infertility: improper function of one of the semen-
following may be added: producing organs, primarily the seminal vesicles
 An equal volume of Dulbecco’s phosphate-buffered
saline Viscosity
 Proteolytic enzymes: a-chymotrypsin, bromelain  The consistency of the fluid: may be related to
specimen liquefaction
 These treatments may affect biochemical tests,  Normal: easily drawn into a pipette and form small,
sperm motility and morphology so their use must discrete droplets that do not appear clumped or
be properly documented stringy when falling by gravity
 Dilution with bromelain must also be accounted for  Droplets forming threads >2cm = highly viscous
when calculating for sperm count and concentration  Reporting: 0 (watery) up to 4 (gel-like) or, low, normal
or high

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pH infections, exposure to toxic chemicals and
 Indicates the balance between the prostatic secretion genetic disorders
and the seminal vesicle secretion  Counts should agree within 10%
 Should be measured within 1h from
ejaculation/collection (loss of CO2)  What should be done if the counts do not
 Normal: 7.2-8.0 (may be detected using pH pas of LIA agree?
or pH testing paper)
 Increased: infection within the reproductive tract
2. Makler: uses undiluted semen; uses heat to
 Decreased: increased prostatic fluid, poorly
immobilize the sperm cells
developed seminal vesicles, ejaculatory duct
 Clinical significance:
obstruction, successful vasectomy
1. Oligospermia – decreased sperm concentration
MICROSCOPIC ANALYSIS (alcoholism, drugs, hypothyroid, renal failure,
gonadotropin deficiency, radiation)
Sperm Concentration 2. Azoospermia – severe oligospermpia or
 Only a single spermatozoon is needed to fertilize an
complete absence of sperm
ovum, but the actual number of sperm present is a 3. Aspermia – no ejaculate
valid measurement of fertility
 Factors that affect sperm concentration: sexual
Sperm Count/Total Sperm Count
 Multiply sperm concentration by the specimen volume
abstinence prior to collection, infection, stress (2-3
 Reference range: >40 M/ejaculate
samples)
 Reference range: 20-250 M/mL *20-160 *>20M 10-
20M = borderline *per mL not per L Sperm Motility
 Presence of sperm capable of forward, progressive
 Counting chambers used:
1. Neubauer: more commonly used; uses dilution
motility is critical for fertility
 Assessed using a well-mixed specimen within 1h from
semen
 Most common dilution: 1:20
collection
 Procedure:
 Diluting fluids:
1. Each laboratory should have a protocol: consistent
a. Sodium bicarbonate and formalin (traditional,
immbolize + preserve cells) amount of semen under same cover slip size
2. Allowed to settle for 1min, then read:
b. Saline
 Percentage of sperm showing actual forward
c. Distilled water (cold tap water)
 Squares used:
movement may be estimated (20 HPFs)
 Examine 200 sperm per slide, count the
a. Four corner and center intermediate squares
(like RBC count): more common percentage of motile categories
 Graded according to both the speed and the
b. Two corner large (WBC) squares *x100,000
*RBC squares: x1M direction of the sperm
 Reference range: >/=50% of sperm with a
 Both sides are loaded and allowed to settle for 3-
5min rating of at least 2.0
 WHO: >/=50% should be a, b, or c; or,
 Counts are performed using bright-field or phase
microscopy >/=25% should be a or b
 Computer-assisted semen analysis
 “Round cells” (immature sperm and WBCs) should
 Determines both velocity and trajectory
not be counted
 Also analyzes sperm concentration and
 Their presence is significant and must be
identified and counted separately morphology
 Manual procedures should be duplicated for
 >1M WBCs/mL –inflammation/infection of
reproductive organs … to infertility accuracy
 Presence of high percentage of immobile sperm and
 >1M spermatids/mL = disruption of
spermatogenesis which may be due to viral sperm clumps require further evaluation (vitamin,
antigen/antibody)

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SPERM MOTILITY GRADING
GRADE WHO RATING DESCRIPTION
4.0 a Rapid, straight motility
3.0 b Slow speed with some lateral movement
2.0 b Slow speed with noticeable lateral movement
1.0 c No forward progression
0 d No movement

WHO ALTERNATE SPERM MOTILITY GRADING

GRADING DESCRIPTION
Progressive motility (PM) Sperm moving with linearity or in a large circle
Non-progressive motility (NP) Sperm moving with an absence of progression
Immotility (IM) No movement

Sperm Morphology 1. Approximately 10L of semen is placed on the slide

 Presence of sperm that are morphologically incapable (and smear as with blood smear)
of fertilization results to infertility 2. Stain using: Papanicolau, Wright’s, Giemsa, or Shorr
 Evaluated with respect to the structure of the head 3. Evaluate at least 200 sperm and report normal and
(penetra), neckpiece, midpiece and tail (motility) abnormal sperm in percentage:
1. Head – oval-shaped; 5 x 3m  Normal: >30% (routine); >14% (Kruger strict
 Acrosomal cap: critical to ovum penetration criteria)
(contains enzymes)  Abnormal: double, giant, amorphous, pin,
 Encompasses ½ of the head and 2/3 of the tapered, constricted heads; double, coiled tails,
nucleus long neckpiece, spermatid (<2%)
2. Neckpiece – attaches the head to the midpiece  Kruger strict criteria:
and the tail  Not routinely performed but recommended by WHO
3. Midpiece - 7m; surrounded by mitochondrial  Measures the head, neck and tail size using stage
sheath for energy production micrometer or morphometry
4. Tail – 45m  Also measures acrosomal cap
 Procedure: evaluated from a thinly smeared, stained  Evaluate the presence of vacuoles
slide under oil immersion  Integral part of CASA

ADDITIONAL TESTING

ABNORMAL RESULT POSSIBLE PROBLEM TEST

Decreased motility with normal count Vitality/viability Eosin-nigrosin stain
Decreased counts Lack of seminal vesicle support medium Fructose level
Mixed agglutination reaction and immunobead test
Decreased motility with clumping Male anti-sperm antibodies
Sperm agglutination with male serum
Normal analysis with continued infertility Female anti-sperm antibodies Sperm agglutination with female serum

Sperm Vitality/Viability (*Necrospermia)  Reference range: >50% *75% living cells (should
 Done when a specimen has normal concentration but correspond with previously evaluated motility)
markedly decreased motility  High number of vital cells but immotile sperm =
 Should be assessed within 1h from ejaculation possible defective flagellum
 Procedure:  High numbers of non-viable and immotile sperm =
1. Specimen is mixed with eosin-nigrosin stain possible epididymal pathology
2. Smear is prepared from the mixture
3. The number of dead cells in 100 sperm are counted Seminal Fluid Fructose
 Living cells: not infiltrated by the dye, remains  Done when low sperm concentration is suspected to
bluish-white be due to lack of seminal vesicle support medium
 Dead cells: infiltrated by the dye, staining red which can be indicated by low or absent fructose level
against a purple background
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 Should be tested within 2h or frozen to prevent 2. Mixed Agglutination Reaction (MAR)
fructolysis  Screening procedure: only detects IgG antibodies
 Fructose screening: resorcinol test (+) orange color  Semen sample with motile sperm is incubated with
 Reference fructose level: >/=13 mol/ejaculate IgG AHG and a suspension of latex particles or
*spectophotometry treated RBCs coated with IgG
 Reference range: <10% of motile sperm
Antisperm Antibodies attached to particles/RBCs (+) microscopic clumps
 May be present in both men and women: detected in 3. Immunobead Test
semen, cervical mucusa or serum  More specific procedure
 Both partners/spouses may demonstrate the  Can detect IgG, IgM and IgA antibodies
antibodies  Demonstrate to which area of the sperm the
 More common: male anti-sperm antibodies antibodies are directed at
 Suspected when clumps of sperm are observed  Sample is mixed with polyacrylamide beads coated
during routine semenalysis with either anti-IgG, anti-IgM or anti-IgA
 Blood-testis barrier: separates sperm from the  Microscopic examination = determination of
male immune system antibodies
 May be disrupted following surgery,  Reference range: presence of beads in <50% of
vasovasostomy, trauma sperm
 Damaged sperm = production of antibodies in the 4. Immunoassay Kits for semen and serum
female partner testing
 Female antibodies: suspected when there is normal
semenalysis with continued infertility Microbial Testing (*Urogenital Infections = 15%
of male infertility >1M WBCs/mL)
TESTS FOR ANTI-SPERM ANTIBODIES  Most frequent site of infection: prostate gland
1. Agglutination Testing  Routine aerobic cultures
 Incubation of fresh, liquefied semen with male  Most common tests: for Chlamydia trachomatis,
serum, female cervical mucosa or serum Mycoplasma hominis, Ureaplasma urelyticum
 (+) agglutination *macroscopic

CHEMICAL TESTING
ANALYTE REFERENCE RANGE ORGAN AFFECTER WITH DECREASED RESULTS
Fructose 13 mole/ejaculate Seminal vesicle
Neutral-A-glucosidase 20 mU/ejaculate
Glycerophosphocholine ? Epididymis
L-carnitine ?
Zinc 2.4 mole/ejaculate
Citric acid 53 mole/ejaculate Prostate
Acid phosphatase 20 units/ejaculate
Glutamyl transpeptidase ?

Post-Vasectomy Semen Analysis  Specimens are routinely tested at monthly intervals

 Only concern is the presence or absence of sperm (do starting 2mos post-vasectomy
not overlook a single sperm)  Continued until two consecutive monthly specimens
show no spermatozoa

SPERM FUNCTION TESTS

TEST DESCRIPTION
Hamster egg penetration Sperm are incubated with species non-specific hamster eggs and penetration is observed microscopically
Cervical mucus penetration Observation of sperm’s ability to penetrate partner’s midcycle cervical mucus
Hypo-osmotic swelling Sperm exposed to low-sodium concentrations are evaluated for membrane integrity and sperm viability
In vitro acrosome reaction Evaluation of the acrosome to produce enxymes essential for ovum penetration

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MEDICO-LEGAL TESTING 4. Florence test: not specific; test for choline
1. Microscopy and fluorescence under UV light: 24h, 3d, 5. Barbiero test: very specific; test for spermine
7d 6. ABO blood grouping
2. Prostatic acid phosphatase 7. DNA analysis
3. Glycoprotein p30 (PSA): more specific; present even
without sperm

CHAPTER 11: SYNOVIAL FLUID ANALYSIS

INTRODUCTION  Commonly collected using syringe that has been
 Synovial Fluid (Joint Fluid) moistened with heparin
 Viscous fluid circulating the movable joints or  Powdered anticoagulants should not be used:
synovial joints (diarthroses) artifacts that may interfere with crystal analysis
 Ultrafiltrate of plasma (concentration similar but are produced
seldom clinically significant) + hyaluronic acid from  Ideally, all testings are done immediately
synoviocytes (viscosity)  Tube Types for Synovial Fluid Tests
 Functions: 1. Microbiology: sterile heparinized or SPS
1. Lubricates joints, reducing friction 2. Hematology: heparin or EDTA
2. Provide nutrients to the vascular-deficient 3. Glucose: sodium fluoride
articular cartilage 4. Chemistry and other tests: nonanticoagulated
3. Lessens shock of joint compression during
activities such as walking or jogging MACROSCOPIC ANALYSIS
 Arthritis
 Collective term for damage of articular membranes Volume
producing pain and stiffness to the joints  Normal volume collected from adult knee cavity:
 Specimen Collection and Handling <3.5mL (sometime, only a few drops: culture, micro)
 Method of collection: arthrocentesis (commonly  May increase to more than 25mL with inflammation
knee)

APPEARANCE SIGNIFICANCE/CORRELATION
Colorless to pale yellow Normal (synovial “ovum” egg white)
Deeper yellow Inflammatory and non-inflammatory effusions
Greenish tinge Bacterial infection
Red Traumatic tap/hemorrhage arthritis (differentiate CSF)
Presence of WBCs
Turbidity
Synovial cell debris and fibrin
Milky Presence of crystals
Macroscopic: resembles rice
*Rice bodies Microscopic: collagen and fibrin
Tuberculosis, septic and rheumatoid arthritis
Macroscopic: ground pepper appearance
*Onchrotic shards
Debris from joint prothesis

Viscosity Normal: ability of the fluid to form a string 4-6cm

–
 Viscous due to the polymerization of the hyaluronic long before breaking
acid which is essential for joint lubrication  Mucin clot/ropes test
 Despite its viscosity, normal synovial fluid don’t clot – Not routinely performed as all forms of arthritis
– Clot formation = presence of fibrinogen cause decreased viscosity
 String test – May be used to identify a questionable fluid as
– Fluid is expressed form the needle 1 drop at a time synovial fluid

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– Measures the amount of the hyaluronate Hypotonic (0.3%) saline if RBCs should be lysed
polymerization Saline with saponin if RBCs should be lysed
– Fluid is added to a solution of 2-5% acetic acid  Normal: <200/L
 Normal/good: solid clot surrounded by clear fluid
 Fair: soft clot RBC Count
 Low: friable clot  Seldom requested
 Poor: no clot  Normal: <2,000/L

HEMATOLOGY/CELL COUNT Differential Count

 Any cell count should be performed immediately or  Should be performed on cytocentrifuged specimens or
refirigerated to prevent cell disintegration on thinly smeared slides
 Very viscous fluid may be pretreated by:  Incubated with hyaluronidase prior to slide
1. Adding one drop of 0.05% hyaluronidase in preparation
phosphate buffer per mL of fluid  Normal WBC differential:
2. Incubating at 37°C  Monocytes and macrophages: ~60%
 Neutrophils: <25%
WBC Count  Lymphocytes: <15%
 Most frequently performed cell count  Eosinophils: <2%
 Uses the Neubauer counting chamber (dilutions just
like for CSF) SYNOVIAL FLUID CELLULAR AND OTHER MICROSCOPIC
 Traditional WBC diluting fluid, acetic acid, cannot be CONSTITUENTS
used, instead:  Primarily mononuclear cells and synovial tissue cells
 Normal saline, may be added with methylene blue

CELL TYPE/INCLUSION MICROSCOPY CORRELATION/SIGNIFICANCE

Bacteria sepsis
Neutrophils Polymorphonuclear leukocytes
Crystal-induced inflammation
Lymphocytes Mononuclear leukocyte Non-septic inflammation
Normal
Monocytes/macrophages Large mononuclear cells, possible vacuolated
Viral infection
Autoimmune diseases
Eosinophils Polymorphonuclear leukocytes with bright orange granules Parasitic infections
Allergic reactions
Similar to macrophage, but may be multinucleated,
Synovial lining/tissue Normal
resembling a mesothelial cell
LE cell Neutrophil containing characteristic ingested “round body” Lupus erythematosus
Reactive arthritis (infection is in another
Reiter cell Vacuolated macrophage with ingested neutrophils
part of the body) *Reit syndrome
Neutrophil with dark, cytoplasmic granules containing Rheumatoid arthritis
RA cell/ragocyte
immune complexes Immunologic inflammation
Cartilage cells Large, multinucleated cells Osteoarthritis
Refractile intracellular and extracellular globules that can Traumatic injury
Fat/lipid droplets
be stained by Sudan dyes Chronic inflammation
Hemosiderin Inclusions within the clusters of synovial cells Pigmented villonodular synovitis
*Rice bodies

CRYSTAL IDENTIFICATION  Ideally performed soon after fluid collection

 Important diagnostic test in the evaluation of arthritis – Ensure crystals not affected by changes in pH, temp
 Causes of crystal formation: – Examined before WBC disintegration as some
 Elevated levels of crystalizing substance: metabolic crystals (MSU and CPPD) can be located intracellular
disorders and/or decreased renal excretion  Fluid is examined as unstained wet prep (like UA)
 Bone and/or cartilage degeneration  May also be observed using Wright-stained smears,
 Injection of medications into a joint (e.g. however, should not replace the use of wet smears
corticosteroids) and crystal polarization
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COMPENSATED SIGNIFICANCE/
CRYSTAL DESCRIPTION OTHER NOTES
POLARIZED LIGHT CORRELATION
Also known as uric acid
Primary crystals seen in synovial fluid
Monosodium urate (MSU) May be extracellular or located within the
Needle-shaped cytoplasm of neutrophils
Negative birefringence Gout
crystals Frequently seen sticking through the
cytoplasm of the cell
Lyse phagosome membranes and therefore
do not appear in vacuoles
Calcium pyrophosphate (CPPD)
Rhombic/squar
Usually located within vacuoles of the
e-shaped, or Positive birefringence Pseudogout
neutrophils
short rods

Cholesterol
Notched,
Negative birefringence Extracellular Associated with chronic inflammation
rhombic plates

Corticosteroid

Flat, variable- Positive and negative

Injection
shaped plates birefringence

Calcium oxalate
Envelopes Negative birefringence Renal dialysis

Calcium phosphate/ apatite Small particles Associated with calcified cartilage

Require degeneration/deposition conditions
No birefringence Osteoarthritis
electron May produce an acute inflammatory
microscopy reaction

CHEMISTRY
 Synovial fluid is chemically an ultrafilrate of plasma, chemistry test values are approximately the same as serum values,
rendering only few chemistry tests to be considered clinically important
 When requested, most test are used using the same methods for serum measurement

ANALYTE DESCRIPTION REFERENCE RANGES

 Most frequently requested test
 Markedly decreased values: inflammatory or septic arthritis
Blood glucose (synovial fluid glucose): 10 mg/dL
Glucose Ex. 80mg/dL – 75mg/dL = _____
 Measured in comparison with blood glucose level after ~8h of fasting
for equilibrium 80mg/dL – 50mg/dL = _____
 Increased levels are found in inflammatory and hemorrhagic disorders <3g/dL
Total protein  Does not significantly contribute to the differential diagnosis of the *~1/3 of serum value as large protein molecules
mentioned disorders are not filtered through the synovial membranes
 Used to confirm diagnosis of gout after an increased serum uric acid
Uric acid Same as blood
levels but monosodium urate crystals were not detected
 Used to monitor the severity and prognosis of rheumatoid arthritis <250mg/dL
Lactate  Provides rapid differentiation between most inflammatory and septic >250mg/dL = septic/rheumatoid arthritis
arthritis *can reach 1,000mg/dL in septic
Acid phosphatase  Used to monitor the severity and prognosis of rheumatoid arthritis ~same as blood

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MICROBIOLOGY AND SEROLOGY – Other than routine culture media, chocolate agar
 Infections occur either secondary to inflammation must also be used as infections caused by
caused by trauma through dissemination of a Haemophilus species and Neisseria gonorrheae are
systemic infection also common
– Gram stain and culture are the two most important  Serologic testing plays an important role as the
tests performed on synovial fluid specimens inflammation process is associated to the immune
– Both tests must be performed on all specimens as system
G/S are only positive in ~50% septic arthritis – Most tests are performed on serum; synovial fluid
cases analyses serve as confirmatory tests to aid in cases
– Bacterial infections are most common; fungal, that are difficult to diagnose
tubercular and viral infection may also occur, thus – Most common serologic tests done to synovial fluid:
special culture procedures must be used when they 1. Autoantibody detection: rheumatoid arthritis and
are suspected lupus erythematosus
– Most common infecting agent: Staphylococcus 2. Antibody detection: Lyme diseases (caused by
aureus followed by Streptococcus pyogenes Borrelia burgdorferi)

CLASSIFICATION, PATHOLOGIC SIGNIFICANCE AND LABORATORY FINDINGS OF JOINT DISORDERS

IIa: IIb:
DISORDER/ I: NON- INFLAMMATORY INFLAMMATORY III: SEPTIC/ IV:
NORMAL
FINDINGS INFLAMMATORY – – CRYSTAL- INFECTIOUS HEMORRHAGIC
IMMUNOLOGIC INDUCED
RA, SLE, RF Trauma, tumors,
scleroderma, hemophilia and
Degenerative
Pathologic polymyositis, Gout and Microbial other coagulation
No joint disorder disorders,
Significance ankylosing pseudogout infection disorder,
osteoarthritis
spondylitis, Lyme anticoagulant
diseases averdose
Clear, pale Clear, Cloudy, Cloudy/milky Cloudy, white to
Color and Clarity Cloudy, red
yellow xanthochromic xanthochromic white yellow-green
Viscosity Good Good Poor Low Variable Low
WBC/L <200 <1,000 2,000-75,000 <100,000 50,000-100,000 Equal to blood
% Neutrophils <25 <30 >50 <70 >75 Equal to blood
RBC presence (–) (–) (–) (–) (+) (+)
Blood-SF Glucose 0-10 0-10 0-40 0-80 20-100 0-20
Difference Normal Normal Slightly decrease Moderate decrease Marked decrease Slight decrease
Possible Presence of Positive G/S
Others – – –
Ab/autoAb cyrstals and/or culture

CHAPTER 12: SEROUS FLUID ANALYSIS

INTRODUCTION –Effusions may result from a disruption of the
 The closed cavities of the body (pleural, pericardial, mechanisms of serous fluid formation and
peritoneal) are each lined by two membranes referred reabsorption or in response to infection and
to as serous membranes namely: inflammatory processes
 Visceral membrane – serous membrane that – Effusions are classified into transudates/exudates
covers the organ within the cavity  Pathological Causes of Effusion
 Parietal membrane – serous membrane that
lines the cavity wall CAUSE EXAMPLE
 The serous fluids are those that fills the space Increased capillary Congestive heart failure
between the two membranes, which provides hydrostatic pressure Salt and fluid retention
Nephrotic syndrome
lubrication as the surface moves against each other Decreased oncotic Hepatic cirrhosis
 Serous is derived from its serum-like composition pressure Malnutrition
 The accumulation of serous fluid is called effusion Protein-losing enteropathy

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Increased capillary
Microbial infection Sterile heparinized: culture, G/S, AFS,
Membrane inflammation cytocentrifuged smear
permeability
Malignancy
 Utility of Analysis:
Malignancy tumors, lymphomas
Lymphatic
Infection and inflammation  Determine the cause of effusion
obstruction  Differentiating transudates from exudative effusion
Thoracic duct injury
 Identify malignancy/infection in exudative effusion
 Collection (needle aspiration):  Establish specific diagnosis and alleviate symptoms
 Pleural fluid: thoracentesis/pleural tap
 Pericardial fluid: pericardiocentesis TRANSUDATES VS EXUDATES
 Peritoneal fluid (ascites): peritoneocentesis  Transudates – occurs during various systemic
 Tubes: disorders that disrupt fluid filtration, fluid
 EDTA: gross appearance, morphology, cell counts reabsorption, or both
and differential count  Exudates – produced by conditions that directly
 Heprinized: chemical, serological, cytological test involve the membrane of the particular cavity
including infection and malignancies

LABORATORY DIFFERENTIATION BETWEEN TRANSUDATES AND EXUDATES

TRANSUDATES EXUDATES
Appearance Clear Cloudy
Color Colorless Depends on condition
Odor Odorless Depends
pH Alkaline Acidic
Specific gravity <0.015 >1.015
Total protein <3 g/dL >3 g/dL
Lactate dehydrogenase <200 IU >200 IU
Cell count <1,000/L WBC, <100,000/L RBC >1,000/L WBC
Spontaneous clotting None Possibly due to fibrinogen
Pleural fluid cholesterol <60 mg/dL >60 mg/dL
Origin Non-inflammatory Inflammatory
Condition CHF, liver cirrhosis, nephrotic syndrome Infection, SLE, malignancy
Glucose As in plasma <plasma level
Chloride 98-106 mEq/L <plasma level
Differential count Few lymphocytes, RBCs, mesothelial cells Lymphocytes, mesothelial cells, eosinophils
Crystals None Plenty: cholesterol, hematoidin
Fluid: serum protein <0.5 >0.5
Fluid: serum LD <0.6 >0.6
Pleural fluid: serum cholesterol ratio <0.3 >0.3
Pleural fluid: serum bilirubin <0.6 >0.6
Serum ascite albumin gradient >1.1 <1.1
Rivalta’s test Negative Positive

PLEURAL FLUID
 Involves the lungs and has a normal volume of <10

PLEURAL FLUID
SIGNIFICANCE
Appearance
Clear, pale yellow Normal
Turbid, white Microbial infection (tuberculosis)
Bloody Hemothorax, hemorrhagic effusion
Chylous material (leakage from the thoracic duct)
Milky
Pseudochylous material (from chronic inflammation)
Brown Rupture from amoebic liver disease
Black Aspergillous
Viscous Malignant mesothelioma (increased hyaluronic acid)
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Differential
Neutrophils (>50%) Pneumonia, pancretitis, pulmonary infarction
Lymphocytes (>50%) Tuberculosis, viral infection, autoimmune disorders
Eosinophils (>10%) Trauma resulting in the presence of air or blood
Normal reactive forms have no clinical significance
Mesothelial cells
Decreased cells or absence are associated with tuberculosis
Plasma cells Tuberculosis
Malignant cells Primary adenocarcinoma and small-cell carcinoma, metastatic carcinoma
Chemistry
<60 mg/dL is considered decreased
Glucose
Low in tuberculosis, rheumatoid inflammation, purulent infections
Lactate Increased in bacterial and tuberculosis infections
Triglyceride Elevated in chylous effusions
Low in tuberculosis, esophageal rupture (6.0)
pH
Decreased in the administration of antibiotics in cases of pneumonia
Amylase Elevated in pancreatitis, esophageal rupture and malignancy
Lactate dehydrogenase Not routinely recommended
CEA Malignancy
CA 125 Ovarian/metastatic uterine cancer
CA 15-3, CA 549 Breast cancer
CYFRA21-1 Lung cancer
Microbiology
Most common organisms Staphylococcus aureus, Mycoplasma tuberculosis
ADA (>40 U/L Highly indicative of tuberculosis; also elevated in malignancy
Other tests ANA and RE

PERICARDIAL FLUID
 Has a normal volume of 10-50 mL

PERICARDIAL FLUID
SIGNIFICANCE
Appearance
Clear, pale yellow Normal, transidate
Blood-streaked Infection, malignancy
Grossly bloody Cardiac puncture, anticoagulation medications
Milky Chylous and pseudochylous material
Differential
Increased neutrophils Bacterial endocarditis
Malignant cells Metastatic carcinoma
Chemistry
Low glucose Bacterial infection, malignancy
CEA Metastatic carcinoma
Microbiology
G/S and culture Bacterial endocarditis
Acid fast stain Tubercular effusion
Adenosine deaminase Tubercular effusion
Most common organisms Hemophilus influenza, Mycoplasma tuberculosis

PERITONEAL FLUID – Recommended over the fluid to serum total protein

 Commonly referred to as “ascitic fluid” and LD ratios for the detection of transudates of
 Ascites – accumulation of fluid in the peritoneal hepatic origin
cavity – Fluid and serum albumin levels are measured
 Has normal volume of <100 mL concurrently and the fluid albumin level is then
 Serum-ascites albumin gradient subtracted from the serum albumin level

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PERITONEAL FLUID
SIGNIFICANCE
Appearance
Clear, pale yellow Normal
Turbid Microbial infection
Green Gallbladder, pancreatic disorders
Blood-streaked Trauma, infection or malignancy
Milky Lymphatic trauma and blockage
Peritoneal lavage >100,000 RBCs/L
Differential
<100,000 RBCs/L Normal
<500 WBCs/L Normal
>500 WBCs/L Bacterial peritonitis, cirrhosis
Neutrophils Increased in bacterial peritonitis
Lymphocytes Predominant cell in tuberculosis
Concentric striations of collagen like material can be seen in benign conditions and also with
Psammoma bodies
ovarian and thyroid malignancies
Malignant cells Malignancy
Chemistry
Glucose Low in tubercular peritonitis, malignancy
Amylase Increased in pancreatic, gastrointestinal perforation, bowel necrosis
Alkaline phosphatase Increased in gastrointestinal perforation or strangulation
BUN/creatinine Ruptured or puncture bladder
Ammonia Bowel strangulation, perforated peptic ulcer, ruptured appendix, ruptured bladder, bowel necrosis
CEA Malignancy of gastrointestinal origin
CA 125 Malignancy of ovarian origin
Microbiology
G/S and culture Bacterial peritonitis
Acid-fast stain Tubercular peritonitis
Adenosine deaminase Tubercular peritonitis
Most common organisms Escherichia coli, Pneumococci

CHAPTER 13: AMNIOTIC FLUID ANALYSIS WITH HCG TESTING

INTRODUCTION  The amount of amniotic fluid increases in quantity
through the pregnancy reaching an approximate peak
Amniotic Fluid of 800-1,200 mL during the third trimester, gradually
 Fills the amnion, the membranous sac that surrounds decreasing prior to delivery
the fetus – First trimester: 35 mL of the fluid comes from
 Functions: maternal circulation
 Provides protective cushion for the fetus – After third trimester, fetal urine is the major
 Allows fetal movement contributor to the amniotic fluid volume
 Stabilizes the temperature to protect the fetus from – Together with urine production, fetal swallowing of
extreme temperature changes the amniotic fluid begins
 Permits proper lung development – Latter third to half of pregnancy
 Exchanges of water and chemicals between the  Polyhydramnios
fluid, the fetus and the maternal circulation – Volume greater than 1,200 mL
 Produces peptides, growth factors and cytokines – Caused by the failure of the fetus to begin
swallowing
Volume – Indication of fetal distress, often associated with
 Regulated by balance between the production of fetal neural tube disorders
urine and lung fluid, and the absorption from fetal
swallowing and intramembranous flow
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– Other associated conditions: fetal structural 2. Genetic predisposition
anomalies, cardiac arrhythmias, congenital a. Family history of chromosome abnormalities,
infections, or chromosomal abnormalities such as trisomy 21 (Down syndrome)
 Oligohydramnios b. Parents carry an abnormal chromosome
– Volume of <800 mL rearrangement
– Caused by increased fetal swallowing, urinary tract c. Parent is a carrier of a metabolic disorder
deformities and membrane leakage d. Family history of genetic diseases such as sickle
– Other associated conditions: congenital cell disease, Tay-Sachs disease, henophilia,
malformation, premature amniotic membrane muscular dystrophy, sickle cell anemia,
rapture, umbilical cord compression resulting in Huntington chorea, and cystic fibrosis
decelerated heart rate and fetal death 3. Problems encountered from previous
pregnancy/pregnancies:
Chemical Composition a. Earlier pregnancy or child with birth defect
 Ultimate source of fluid water and solutes is the b. Previous child with a neural tube disorder such
placenta as spina bifida, or ventral wall defects
 Composition is similar to maternal plasma with a (gastroschisis)
small amount of sloughed fetal cells from the skin, c. Three or more miscarriages
digestive system and urinary tract (may be used for 4. Abnormal maternal serum tests: Down’s NTD
cytogenetic analysis) a. Alpha-fetoprotein Dec Inc
 Amniotic fluid creatinine – measured to determine b. Human chorionic gonadotrophin Inc
fetal age c. Unconjugated estriol Dec
 1.5-2.0 mg/dL – <36wk of gestation d. Inhibin A Inc
 >2.0 mg/dL - >36wk of gestation  Later in the pregnancy (20-42wk) to evaluate:
1. Fetal lung maturity
MATERNAL URINE VS AMNIOTIC FLUID 2. Fetal distress
PARAMETER MATERNAL URINE AMNIOTIC FLUID 3. HDN caused by Rh blood type incompatibility
Creatinine 10 mg/dL <3.5 mg/dL 4. Infection
Urea 300 mg/dL <30 mg/dL
Glucose Less common More common
Protein Less common More common
Specimen Collection
 Amniocentesis is the collection of amniotic fluid by

 Fern Test needle aspiration into the amniotic sac using

 Also differentiates maternal urine versus amniotic
continuous ultrasound for guidance
 Safety is most assured if collection is performed
fluid
 Test used to evaluate premature membrane
beyond the 14thwk of gestation
 Methods of Collection:
ruptures
1. Transabdominal – most frequently performed
 Vaginal specimen is spread on a glass slide 
method
complete air drying at room temperature  2. Vaginal – greater risk for infection
observed microscopically: fern-like crystals due to  Maximum of 30mL is collected in sterile syringes
protein and sodium chloride ([+] screen for – First 2-3 mL is discarded: possible contamination
amniotic) with maternal blood, tissue fluid and cells
– Specimens are transferred to plastic containers
SPECIMEN COLLECTION, HANDLING AND (cells tend to adhere on glass containers)
PROCESSING
Specimen Handling and Processing
Indications for Performing Amniocentesis  All handling procedures should be performed
 15-18wk gestation for the following conditions to
immediately and the specimen delivered promptly to
determine early treatment or intervention: the laboratory
1. Mother’s age of 35 or older at delivery

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TEST FOR PROPER HANDLING AND PROCESSING
Fetal lung maturity tests Placed in ice for delivery to the laboratory and kept refrigerated
Immediately protected from light by placing the specimens in amber-colored tubes, wrapping
Bilirubin tests (detection of hemolytic
the collection tube in foil, or by use of a black plastic cover for the specimen container
disease of the fetus and the newborn)
Markedly decreased values may be obtained with as little as 30min exposure to light
Cytogenetic studies and microbial studies Must be processed aseptically and maintained at room temperature or body temperature
Separated from cellular elements and debris immediately to prevent alteration of chemical
Chemical analysis (including bilirubin)
constituents by cellular metabolism or disintegration

MACROSCOPIC ANALYSIS
APPEARANCE SIGNIFICANCE/CORRELATION
Colorless with slight to moderate turbidity Normal (due to cellular debris)
Traumatic tap, abdominal trauma, intra-amniotic hemorrhage
Blood-streaked
(different maternal/fetal blood: Kleihauer-Betke
Yellow Bilirubin (hemolytic disease of newborn)
Dark green Meconium; may be present due to fetal distress
Dark red-brown Fetal death

TESTS FOR FETAL DISTRESS  Interferences:

Meconium = falsely low A450, not acceptable for
Hemolytic Disease of the Fetus and the spectrophotometric analysis
Newborn  Oxyhemoglobin = maximum absorbance of
 The oldest routinely performed laboratory test in hemoglobin at 410 nm; extraction with chloroform
amniotic fluid is for the evaluation of fetal anemia  Control: 1:10 dilution of normal saline
due to the hemolytic disease of the fetus and the
newborn
 Incidence of this disease has been decreasing rapidly
since the development of methods to prevent anti-Rh
antibody production in Rh-negative post-partum
mothers but not for other antigens
 Commonly caused by anti-Rh antibodies of Rh-
negative mothers which causes the destruction of
fetal RBCs when the antibodies cross the placenta;
other antibodies to fetal antigens also
 The destruction of the fetal RBCs results in the
appearance of the RBC degradation product,
unconjugated bilirubin in the amniotic fluid
 By measuring the amount of bilirubin in the fluid, the
extent of hemolysis taking place may be determined;
the danger this anemia presents to fetus may assess

Bilirubin Analysis (Optical Density [OD] 450)

 Measured by spectrophotometric analysis using serial Neural Tube Defects
dilutions in intervals between 365 and 550 nm 1. Alpha-fetoprotein
 Normal fluid: OD is highest at 365 nm and  Produced by the fetal liver prior to 18wk gestation
decreasing linearly to 550 nm (straight line)   levels in maternal blood and amniotic fluid occurs
 Presence of bilirubin: there is rise seen at OD 450 when the skin fails to close over the neural tissue
(wavelength of maximum bilirubin absorption  Indication to measure amniotic fluid AFP:”
 Difference between the baseline and the OD 450 increased maternal serum AFP, family history of
(A450) = bilirubin concentration  Normal values based on weeks of gestation
 Plotted on a Liley graph to determine the severity (maximum AFP 12-15wk)
of the hemolytic disease (>0.25) 2. Acetylcholinesterase
 Confirmatory test

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 Not performed on a bloody specimen –Phosphatidyl glycerol is another lung surface
lipid that is also essential for adequate lung
TESTS FOR FETAL MATURITY (FETAL LUNG maturity which can only be detected after the
MATURITY) 35thwk of gestation (delayed – GDM mother)
 Respiratory distress syndrome – the most common – Lamellar bodies densely packed layers of
complication of early delivery and a cause of phospholipids that represent a storage form of
morbididty and mortality in the premature infant pulmonary surfactant
 Due to the lack of lung surfactant, which keeps the  Secreted by Type II pneumocytes at about the
alveoli open during inhaling and exhaling 24thwk pf gestation
th
 Surfactant decreases the surface tension on the  Enter the amniotic fluid at about the 26 wk of
alveoli, so they can inflate more easily gestation, increasing from 50,000-200,000/L
– Lecithin is the primary component of the lung by the end of the third trimester
surfactant which accounts for alveolar stability;  Phospholipid Levels during Gestation:
increased production occurs after the 35thwk th
 Up to 26 wk of gestation: lecithin <
– Sphingomyelin is produced at a constant rate sphingomyelin
after the 26thwk and serves as a control for the th
 36 wk of gestation: lecithin = sphingomyelin
th
rise in lecithin  Beyond 36 wk: lecithin > sphingomyelin

TEST DESCRIPTION/NOTES RESULT FOR FETAL LUNG MATURITY

Lecithin/sphingomyelin Reference method for fetal lung maturity
ratio Blood or meconium cause falsely elevated result 2.0
Immunologic test for phosphotidyl glycerol
Amniostat-FLM May be used instead of L/S ratio for specimens with blood/ Agglutionation (slight higher false–)
meconium (TLC of L/S/PG for more accurate FLM measurement)
AF + 95% ethanol  shake for 15s  stand for 15min Continuous line of bubbles around the
Foam shake test
(alcohol antifoaming agent) outside edge
Modification of the foam test
0.5 mL AF + increasing amounts of 95% ethanol
Foam stability index >47 (good correlation with L/S and PG)
(0.42-0.55 mL in 0.01 mL increments)
Cannot be used if AF is contaminated with blood.meconium
Increased phospholipids = decreased microviscosity
Microviscosity
Measured using fluorescence polarization assay 55 mg/g
Uses the platelet channel of automated hematology analyzers
Lamellar body count Specimens with blood, meconium and mucucs should not used 32,000/mL
(lamellar body diameter is similar to plts)
O.D. 650 Increased lamellar bodies = increased optical density 0.150

HUMAN CHORIONIC GONADOTROPHIN TESTING  Peaks 100,000-200,000 mlU/mL after 60-80d of

fertilization, and at the 15th-16thwk, declines to a level
Human Chorionic Gonadotrophin maintained through the rest of the pregnancy
 Produced by syncytiotropholast of the placenta during  Regresses to nondetectable level within 2wk post-
pregnancy for the rescuing and maintaining corpus partum
luteum beyond its normal 14-d lifespan
 Is a glycoprotein composed of two subunits: Ectopic Pregnancy
1. Alpha-subunit: aidentical to LH, FSH and TSH  Caused by: pelvic inflammatory disease, microsurgical
2. Beta-subunit: almost similar to LH but the techniques for treatment of fallopian tube disease,
additional 30 amino acid portions confers the and the increased use of ovulation induction
specificity to HCG medications
 Diagnosis:
Normal Pregnancy B-hCG: 1. Doubling time does not occur
 Detectable as early as 6-8d after fertilization 2. Serum B-hCG does not rise at least 66% within 48h
 Level doubles approximately every 2d until (80% sensitivity)

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3. Serial measurements: 2/3 of the patients exhibit  Test: most common sample is urine; serum/plasma is
decrease, plateau or inability to achieve the normal also used
slope for the production of B-hCG
Immunologic Tests to Detect Beta-hCG
Abortion and Other Conditions Related to B-hCG 1. Direct/Agglutination – preferred specimen: early
1. Spontaneous abortion – serum B-hCG becomes morning specimen
undetectable within 9-36d (median: 19d) 2. Indirect/Agglutination Inhibition – urine +
2. Induced abortion – serum B-hCG become anti-hCG  +hCG coated RBCs/latex
undetectable within 16-60d (median: 30d) 3. Radioimmunoassay – competitive binding to anti-
3. Hyatidiform mole/Gestational Trophoblastic hCG, hCG from px vs radiolabeled hCG
Disease (aberration of pregnancy; becomes a tumor) 4. Immunoradiometric – radiolabeled anti-hCG;
 Enlarged uterus with (+) B-hCG but without fetal second antibody on solid phase
heartbeat 5. Immunoenzymatic/Enzyme immunoassay –
 Pelvic sonogram: characteristic snowstorm pattern common in both home and lab test kits antibody on
4. Testicular cancer – serum B-hCG in conjuction wAFP solid phase and Ab with indicator enxyme; color
5. Ovarian germ cell tumor – (+) serum B-hCG but intenstity = prop
without pregnancy

BIOASSAYS TO DETECT BETA-hCG

TEST ANIMAL USED AREA OF INJECTION (SAMPLE) POSITIVE RESULT
Hogben Female toad Lymph sac/*thigh muscle (urine) Oogenesis/extrusion of eggs
Galli-Mainini Male frog Subcutaneous/*thigh muscle (urine/serum) Spermatogenesis/sperm in urine after 30min
Friedmann Corpora lutea/hemorrhagica (red spots on
Virgin female rabbit Marginal ear vein (urine)
Hoffmann ovary)
Immature female Corpora lutea/hemorrhagica (ovarian
Ascheim-Zondek
mice hyperemia)
Subcutaneous (urine)
Frank-Berman Immature female rat
Kelso Ovarian hyperemia
Female virgin rat
Kupperman Intraperitoneal (urine)

CHAPTER 14: GASTRIC FLUID ANALYSIS WITH SWEAT TESTING

GASTRIC FLUID  Gastric acid secretion in response to insulin-
 Contains hydrochloric acid from the parietal cells and induced hypoglycemia
pepsin (activated pepsinogen) from the chief cells  Terminologies regarding gastric acidity
– Euchlorhydia – normal free HCl
Functions – Hyperchlorhydia – increased >60 mEg/L,
1. Determination of gastric acidity: peptic ulcer
 Diagnosis and monitoring of pernicious anemia and – Hypochlorhydia – decreased, >3.5 pH but fall
ulcer (peptic vs duodenal) after stim, gastric ulcer/CA, chronic gastritis
 Identify proper surgical procedure for peptic ulcer – Achlorhydia – absence, >3.5 pH does not fall,
treatment PA, advanced gastric ulcer, pellagra niacin
 Support of hypersecretory state characteristic of deficiency
Zollinger-Ellison syndrome – Anacidity – failure to produce pH <6.0 after
 Rare disorder exhibited by the increased levels stimulation, PA
of gastrin produced by a pancreatic tumor or 2. Diagnosis of other gastric diseases
enlarged pancreas causing the excessive 3. Culture for the recovery of Mycobacterium
secretion of gastric juice tuberculosis
 Determination of the completeness of vagotomy 4. Toxicology

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Composition (99% water) 3. Rest, relaxed and tube should not be resisted
1. Hydrochloric acid: pepsinogen  pepsin; limited 4. Test meal after first collection
hydrolysis of polypeptides and disaccharides  Types of specimen: basal acid output and maximum
2. Electrolytes: all electrolytes in body fluids acid output
3. Mucus: mucoproteins and mucopolysaccharides;  Duration of collection:
protection from autodigestion – 1-h collection: four 15-min specimens or single 1-h
4. Enzymes specimen
a. Pepsin: major digestive enzyme, activate at optimal – 2-h collection: for insulin hypoglycemia test
pH 1.6-2.4; 3.6 protein  peptones, protea  Proper position: sitting/lying on left side and head
b. Gastricsin: proteolysis elevated 45°
c. Rennin: weak proteolysis, mlik curdling  Gastric tubes:
d. Gastric lipase: 4.5-5.5, important in neonates 1. Levine – gastric collection; rubber; has the
(underdeveloped pancreas) and SF smallest diameter; inserted through the nose
e. Non-digestive enzymes: LDH, AST, ALT, etc 2. Rehfuss – for gastric and duodenal contents;
5. Other substances inserted through the mouth
a. Proteins: albumin and gamma-glbulins 3. Sawyer – gastric collection; longest tube
b. Intrinsic factor 4. Ewald or Boa – for washing/emptying of stomach
in cases of poisoning
Specimen Collection and Handling  Causes of failure of the gastric fluid to flow:
 Method of collection: aspiration 1. Tube has not reached the stomach
 Preparation: 2. Introduction of tube into the wrong place (e.g.
1. 12h fasting, no medications for 24h trachea)
2. Instructed not to swallow excessive saliva during 3. Clogged tube (with food particles or mucus)
collection 4. Completely empty stomach due to hypermotility

GASTRIC STIMULANTS
STIMULANT CONTENT PURPOSE
Test Meals
Ewald’s (Breakfast) Bread (no butter), weak tea or water (no sugar) Routine for gastric analysis
Boa’s Oatmeal (with water, and a pinch of salt) Lactic acid detection (L. bulgaricus)
Riegel Beef steak, mashed potato, Bouillon soup Achylia and hypoacidity
Heckman Egg albumin, water, methylene blue *Introduced through a tube
Lavine Ethanol and methylene blue (blue  green [alkaline]) Detects reflux from duodenum
Sham feeding Chewing sandwich without swallowing Detects completeness of vagotomy
Chemical stimulants (better)
Pentagastrin Method of choice (resembles gastrin); do not cause patient discomfort
Insulin (hypoglycemia) Injected intravenously; tests the completeness of vagotomy
Injected subcutaneously; cuases stomachache, bradycardia, flushing of face, headache and
Histamine
lacrimation; used for the detection of PA (true achlorhydia)
Histalog Histamine with lower concentration

MACROSCOPIC ANALYSIS SIGNIFICANCE/CORRELATION

Volume
20-50 mL Normal (fasting specimen)
20-60 mL up to 120 mL After test meals
45-150 mL After chemical stimulants
Pernicious anemia
<20 mL Chronic alcoholism
Stomach: hypermotility, leather-bottle stomach
Zollinger-Ellison syndrome
>50 mL Pyloric (ulcer or extreme intestinal obstruction
Stomach: hypomotility, chronic dilatation, syphilis
Appearance
Colorless to pale gray with mucus Normal

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Yellow-green Large amount of vile (fresh y old g)
Red Fresh blood
Coffee ground Old blood (food particles = obstruction)
Odor
Odorless, slightly sour or faintly pungent Normal
Fecal odor Intestinal obstruction (causing regurgitation)
Foul or putrid odor Necrotic lesion, cancerous ulcer, malignant stenosis
Ammoniacal odor Uremia
Rancid odor Butyric and lactic indicating stenosis and fermentation

CHEMICAL ANALYSIS Lactic Acid

 Normally absent presence is indicative of cancer
pH  Tests: all uses ferric chloride
 1.6-1.8 1. Modified Uffelman’s – also uses phenol; color:
yello
Total Acidity 2. Strauss – also uses ether; color: yellow/green
 Free HCl and other acids (acid salts, lactic acid, butyric 3. Kelling’s – color: yellow
acid, amino acids)
 Normal: 40-70 mEq/L Occult Blood
 Tests:  Normally absent (small amount: trauma)
1. Phenolphthalein – uses alcoholic  Large amounts are abnormal: peptic ulcer, gastric CA,
phenolphthalein; color: deep pink prolonged vomiting, bleeding papilloma, stomach or
2. Toopfer’s Test – uses spdum hydroxide and esophageal varices
phenolphthalein; color: salmon pink  especially old bile: duodenal obstruction or ulcer, rigid
pylorous due to stenosis or adhesions,
Free HCl hyperchlorhydia, gall-tract disease
 Normal: 20-40 mEq/L  Hematemesis vs. hemoptysis (dark red/brown due to
 Tests: acid vs. bright red, frothy, mucus, alkaline)
1. Topfer’s – uses sodium hydroxide; color: yellow  Test: benzidine or guiac
2. Boa’s – uses resorcinol and cane sugar alcohol;
color: rose red Bile
3. Gunzberg’s – uses phloroglucin, vanillin and 95%  Normally absent (small amount: strain when tube is in
ethanol; color: purplish red stomach)
4. Dimethylaminobenzol – color: cherry red  Large amounts are abnormal, especially old bile:
5. Diagnex Tubeless Test – ion exchange resin duodenal obstruction or ulcer, rigid pylorous due to
(amberlite cation) coupled w/azure A or azure blue stenosis or adhesions, hyperchlorhydia, gall-tract
is given by mouth; presence of free HCl causes the disease
release of the dye which is then rapidly abdorbed  Hematemesis vs. hemoptysis (dark red/brown due to
by the intestines, into the blood and excreted in acid vs. bright red, frothy, mucus, alkaline)
urine (caffeine stimulation); specimen: urine  Test: benzidine or guiac

GASTRIC ACIDITY RESULTS

SIGNIFICANCE BASAL ACID OUTPUT (mEq/h) MAXIMAL ACID OUTPUT (mEq/h) BAO/MAO
Normal 2.5 25.0 10%
Duodenal 5.0 30.0 17%
Zollinger-Ellison syndrome 18.0 25.0 72%
Pernicious anemia 0 0 0

MICROSCOPIC ANALYSIS 2. Epithelial cells

1. Mucus  Squamous cells: from esophagus; clinically
 Normal insignificant
 Excessive due hypertrophic gastritis, ulcer/cancer  Columnar cells: gall bladder disease

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3. Tissue fragments  Mycobacteriuim tuberculosis – pulmonary and
 Small amount is normal intestinal tuberculosis
 Seen in cancer and cancerous infiltration, necrotic 8. Parasites – Giardia lamblia
tissue 9. Food residue – seen in in stasis and pyloric
4. RBCs obstruction (stain blue in Lugol’s iodine = starch)
 Not normally present
 Seen in irritation of gastric mucosa by the tube, SWEAT TESTING
trauma, ulcer, cancer or erosion  Primarily used to diagnose cystic fibrosis
5. WBCs (mucoviscidosis): an autosomal recessive metabolic
 Not normally present disorder affecting the mucus-secreting glands of the
 Seen in gastritis and gastric cancer body, as well as causing pancreatic insufficiency,
6. Yeast cells respiratory distress and intestinal obstruction
 Normally found in few numbers  Method of sweat collection: Gibson-Cook pilocarpine
 Increased number is indicative of retention of food iontophoresis (with mild current)
and fermentation  Method of testing:
7. Bacteria  Sodium: FEP or ISE
 Sarcina spp. – seen in ulcer-producing pyloric  Chloride: titration
obstruction; presence exclude cancer  Values/results:
 Lactobacillus acidophilus – a.k.a. Boas-Oppler  Normal: up to a maximum of 40 mEq/L
bacillus, seen in obstruction-causing cancer  Diagnostic of CF: >70 mEq/L (>60 mEq/L)
indicating achlorhydia with stagnation

CHAPTER 15: SPUTUM ANALYSIS WITH BRONCHOALVEOLAR LAVAGE

INTRODUCTION

SPUTUM VS SALIVA
SPUTUM SALIVA
 Viscous to elastic tracheobronchial secretions with added
 Thin, clear, watery and slightly viscous fluid that is a
cellular exfoliations, and possible saliva and normal oral flora
mixed product of secretions from three pairs of salivary
 Differentiated from pure saliva by the presence of dust cells or
glands: parotid, submaxillary and sublingual glands
carbon-laden macrophages

Functions of Analysis b. Sputum induction – for non-cooperative patients

1. Diagnosis of respiratory conditions: primary utility i. Aerosols: 10% NaCl or sterile distilled water
 Malignancies of lungs and other respiratory organs ii. 10% prophyleneglycol in saline solvent
 Infections (i.e. tuberculosis) c. 24-h – for volume measurement
 Inflammations (i.e. pulmonary lesion) 2. Throat swab – for babies
 Other respiratory disease (i.e. asthma) 3. Tracheal aspiration – for severely debilitated
2. Aids in the selection of appropriate therapy for a patients
specific disease  Preservation:
1. Refrigeration – preserves tubercle bacilli
Specimen Collection and Handling  Some sources indicate that tubercle bacilli are
 Method of collection: also preserved at room temperature
1. Expectoration 2. Formalin – kills the bacteria
a. First morning – most concentrated, most routine,
most preferred

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MACROSCOPIC ANALYSIS
SIGNIFICANCE/CORRELATION
Odor
Foul/putrid Anaerobic infections (i.e. gangrene), advanced necrotizing tumors/lesions, tuberculosis, lung abscess
Sweetish Bronchiectasis, bronchomoniliasis, tuberculosis
Fecaloid Necrosis, liver abscess, enteric bacterial infection, rupturing below the diaphragm
Cheesy Necrosis, tumors, empyema
Volume
Increased Bronchiectasis, lung abscess, edema, gangrene, tuberculosis, pulmonary hemorrhage
Decreased Acute bronchitis, bronchial asthma, early pneumonia, healing of lung disease
Consistency
Watery Normal, made up of mucus only
Serous/frothy Edema
Mucoid Asthma, acute bronchitis, whooping cough
Mucopurulent or purulent Bronchiectasis, pneumonia, lung abscess, gangrene, tuberculosis
Appearance
Colorless and translucent to
Normal, made up of mucus only
white or faint yellow
Gray Presence of pus/epithelial cells
Opaque white or yellow Presence of pus/epithelial cells: tuberculosis, chronic bronchitis, lobar pneumonia
Breakdown of neutrophils (verdoperoxidase), Psuedomonas aeroginosa infection, presence of bile,
Bright green
rupture of liver
Fresh blood: hemorrhage
Red or bright red
Blood streaks: tuberculosis, bronchiectasis
Anchovy sauce or rusty Old blood: lobar pneumonia (with pus), tuberculosis, gangrene, hemorrhage from lung due to
brown infarction
Rusty brown/brown w/o pus Congestive heart failure
Currant, jelly-like Klebsiella pneumoniae infection
Prune juice Pneumonia, chronic lung cancer
Olive green/grass green Chronic cancer
Black Inhalation of dust, dirt, carbon, charcoal; heavy smokers; anthrax

MACROSCOPIC STRUCTURES
STRUCTURES DESCRIPTION SIGNIFICANCE/CORRELATION
1st (top) = frothy mucus
Formation of layers 2nd (middle) = opaque, water material Bronchiectasis, lung abscess, gangrene
3rd (bottom) = pus, bacteria, tissues
White or gray branching tree-like casts of the bronchi
Bronchial casts Lobar pneumonia, bronchitis, diphtheria
Mainly made up of fibrin
Cheesy masses Pinpoint to pea-size fragments of necrotic poulmonary tissues Gangrene, tuberculosis, lung abscess
White to yellow, waxy, coiled mucus strands
Consisting of epithelial cells (mainly eosinophils), sometimes
Cruschman’s spirals Bronchial asthma, bronchitis, tuberculosis
with Charcot-Leyden crystals
May also be observed microscopically
Yellow or gray, pinhead to bean-size causeous material that
Dittrich’s plugs Bronchitis, bronchiectasis, bronchial asthma
causes foul odor when crushed
Lung stones/ broncholiths/ Yellow/white/gray calcification/ fragments of infected and
Chronic tuberculosis, histoplasmosis
pneumoliths necrotic pulmonary tissue
Aspergilloma Rounded masses of fungal debris A. fumigatus infection

MICROSCOPIC ANALYSIS
STRUCTURES DESCRIPTION SIGNIFICANCE/CORRELATION
Slender fibrils with double contour and curled ends from
Elastic fibers Denotes destructive lung disease (i.e. tuberculosis)
the walls of alveoli and bronchioles
Clusters of vacuolated columnar epithelial cells with
Creola bodies Bronchial asthma
ciliated borders
Myelin globules Colorless globules occurring in variety of sizes and No clinical significance; mistaken as biastomyces
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bizarre forms
Crystals
Colorless, hexagonal, double pyramid, often needle-like
Charcot-Leyden crystals bodies arising from eosinophils degradation Bronchial asthma
Stains black with hematoxylin and red with eosin
Hematoidin Rhombic-shaped crystals Pulmonary infarction, lung abscess
Cholesterol Broken-edge; stair-cause; notched plate
Lung abscess
Fatty acids Colorless needle-like cyrstals
Pigmented cells
Carbon-laden cells Macrophages containing black granules Smoke inhalation, heavy smokers, anthrax
Siderocytes/heart failure cells Macrophages containing hemosiderin Congestive heart failure
Asbestos
Fungi A. fumigatoss, C. albicans, C. neoformans, B. dermatitidis, P. brasiliensis, C. immitis, H. caosulatum
Parasites Migrating larvae (ASH), T. canis, E. granulosus, P.westermani, E. gingivalis, E. histolytica, T. tena
Others Neoplastic cells, bacteria leukocytes

BRONCHOALVEOLAR LAVAGE
 Method of obtaining cellular and microbiologic information from the lower respiratory tract (alveoli) using a
bronchoscope through which saline is instilled into the distal bronchi and then withdrawn
 Important diagnostic test for P. jiroveci (formerly P. carinii) infection

PERCENTAGE CELLS SIGNIFICANE/CORRELATION

56-80% Macrophages (dust cells) Normally predominant
1-15% Lymphocytes Interstitial disease, pulmonary lymphoma, nonbacterial inf
<3% Neutrophils Cigarettes smoking, bronchopneumonia, toxin exposure
0-2% Eosinophils Hypersensitivity and allergic reactions, parasitic infections
4-17% Ciliated columnar epithelial cells normal

CHAPTER 16: FECAL ANALYSIS

INTRODUCTION  Approximately 9,000 mL of fluids enter the digestive
tract, under normal conditions, only 500-1,500 mL
Physiology reaches the large intestines (7,500-8,500 reabsorbed
 Fecal specimens by SI)
 Contains bacteria, cellulose and other undigested  In normal state, only 150 mL of water is excreted in
foodstuffs, gastrointestinal secretions, bile the feces
pigments, cell form intestinal walls, electrolytes  Absorbing capacity of the large intestine: 3,000 mL
and water
 Approximately 100-200 g of feces is passed per day Specimen Collection and Handling
 Associated with strong odor due to bacterial  Types of Sample
metabolism and intestinal gas (flatus), 75% water, 1. Random specimen (small amounts) – for qualitative
25% solid testing and microscopic exam RBCs, WBCs, fats,
 Large amount of flatus: fiber
carbohydrates/oligosaccharides resistant to 2. Timed specimens – for quantitative testing
digestion and lactose-intolerant consume milk – 3-d collection – most representative sample
 Small intestine – primary site pf the final breakdown  Preservation
and absorption of proteins, carbohydrates and fats 1. Refrigeration
– Pancreas – secretes trypsin, chymotrypsin, amino 2. Chemical – 2-10% formalin, 95% ethanol, glycerol
peptidase, and lipase (amylase) in NSS, etc.
– Liver – bile salts (deficiency of any = inability to
digest and abs) MACROSCOPIC SCREENING
 Quantity: 100-200 g/d

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SIGNIFICANCE/CORRELATION
Appearance
Light to dark brown Normal (urobilin/stercobilin)
Black, tarry (melena) Upper GI bleeding, iron ingestion charcoal, bismuth (antacids)
Red (hematochezia) Lower GI bleeding, beets and food coloring, rifampin
Pale yellow, white, gray (acholic) Bile duct obstruction, pancreatic disease, barium sulfate
Yellow Milk diet, corn meal, rhubarb, fats
Green Biliverdin, oral antibiotics, green vegetables, food coloring
Blue Prussian blue, grape soda
Violet/purple Porphyria
Consistency
Bulky/frothy/greasy may float Bile duct obstruction, pancreatic disorder, stratorrhea
Butter-like Cystic fibrosis
Blood-streaked mucus Colitis, dysentery (bacterial or amoebic), malignancy, damaged intestinal walls
Mucus-coated Intestinal inflammation, irritation
Ribbon-like/slender/flattened Intestinal constriction
Rice watery Cholera (Vibrio cholera)
Pea-soup Typhoid (Salmonella typhi)
Small hard/scybalous (goat droppings) Constipation
Odor
Foul to offensive Normal (due to skatole, indole and butyric acid)
Putrid Ulcerated and malignant tumors of the lower bowel
Extremely foul Undigested proteins, bacterial contamination
Sour, rancid Gas formation, fermentation of carbohydrates, unabsorbed fatty acids
Rotten egg odor Steatorrhea

BRISTOL STOOL CHART disorders (Zollinger-Ellison syndrome,

TYPE DESCRIPTION hyperthyroidism), neoplasms, and collagen
1 Separate hard lumps, like nuts (hard to pass) vascular disease
2 Sausage-shaped but lumpy  Substances: drugs (caffeine, stimulant laxatives),
3 Sausage with cracks on surface
4 Sausage, snake smooth surface
hormones
5 Soft blobs with clear cut edge 2. Osmotic – retention of water and electrolytes due
6 Fluffy with ragged ends (mushy) to incomplete breakdown or reabsorption of food;
7 Watery seen with increased osmotic gap caused by un-
absorbable solutes which increases stool osmolality
DIARRHEA and low electrolytes concentration
 Defined as an increase in daily stool weight above  Caused by maldigestion and malabsorption
200 g, increased in the liquidity of the stools, and diseases
frequency of more than three times per day  Lactose intolerance (and other disaccharide
 Four factors for classification: illness duration, deficiency), celiac sprue, amoebiasis
mechanism, severity and stool characteristics  Laxatives, magnesium-containing antacids,
 Illness duration: acute: <4wk, chronic: >4wk antibiotics administration
 Major mechanisms:
1. Secretory – increased of water and electrolytes DIFFERENTIAL TESTS FOR SECRETORY AND
by the intestine which overrides the reabsorptive OSMOTIC DIARRHEA
ability of the large intestines SECRETORY OSMOTIC
 Bacterial (enterotoxins: E.coli, Salmonella, Osmotic gap <50 mOsm/kg >50 mOsm/kg
Shigella, Vibrio cholera, Campylobacter, Stool sodium >90 mmol/L <60 mmol/L
Fecal output >200 g/24h <200 g/24h
Staphylococcus), viral and parasitic pH >5.6 <5.3
(Crysptosporidium) infections Reducing substances Negative Positive
 Non-communicable diseases: inflammatory
bowel disease (Crohn disease, ulcerative colitis, 3. Altered motility – enhanced motility or slow
lymphocytic colitis, diverculitis), endocrine motility which are seen in irritable Bowel Syndrome
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 Enteritis, complications of malabsorptionm rapid intestinal resection, celiac disease [most common
gastric emptying (RGE) dumping syndrome cause of malabsorption in dev con], tropical sprue,
 Parasympathetic drugs lymphoma, Whipple disease, Giardia lamblia
infection, intestinal ischemia)
STEATORRHEA  Screening test: qualitative fecal fats (microscopic
 Defined as an increase in stool fat that exceeds 6 g/d exam of feces for fat globules); also used to monitor
 Useful in diagnosing pancreatic insufficiency, other the treatment of patients for malabsorption disorders
pancreatic disorders and small-bowel disorders – Uses dyes like Sudan III (most routine), Sudan IV or
causing malabsorption (bacterial overgrowth, oil red O: neutral fats and split fats

NEUTRAL FATS VS SPLIT FATS

NEUTRAL FATS SPLIT FATS
 Better indicator then neutral fats
Description  Readily stained by Sudan III
 Both number and size of the fat droplets are considered
 Stool suspension (1 part stool + 2 parts  Emulsified stool + drop 36% acetic acid + 2 drops dye
water) + 1 drop 95% ethanol + 2 drops dye then apply cover slip + heat (ch)
Preparation
 Orange droplets = neutral fats/triglycerides  Orange droplets = split fats/fatty acids, fatty acid
 Steatorrhea: 60 droplet/HPF salts, cholesterol
 Normal: 100 droplets (<4m)
Results  Slightly increased: 100 droplets (1-8m)
 Increased: 100 droplets (6-75m) = steartorrhea
Malabsorption Normal Increased
Maldigestion Increased Normal or increased

 D-xylose test – another test to differentiate 2. Gravimetric method (detects 100% of fat)
malabsorption from maldigestion 3. Hydrogen nuclear magnetic resonance
 Specimen: urine spectroscopy
 Normal D-xylose = maldigestion; low D-xylose = 4. Acid steatocrit
malabsorption  Reliable too to monitor patient’s response to
 Trypsin – historically tested using the x-ray therapy
film/gelatin test (emulsified s + film – trypsin   May be used to screen steatorrhea in pediatric
clearing) patients
 Chymotrypsin – may also be tested using gelatin 5. Near-infrared reflectance spectroscopy
test but commonly measured using  48-72h spectroscopy with special software
spectrophotometry
 Elastase I CREATORRHEA
– Detected using ELISA requiring only a single stool  Defined as an abnormal excretion of undigested
sample muscle fibers in feces
– Pancreas specific, present in high concentrations  Microscopic detection aids in the diagnosis and
and very resistant to degradation monitoring of patients with pancreatic insufficiency
– Specific in differentiating pancreatic from non- – Also seen in biliary obstruction and gastrocolic
pancreatic causes in patients with steatorrhea fistulas
 Confirmatory test: quantitative fecal test  Frequently ordered in conjunction with fecal fats
– Requires collection of at least 3-d specimen; fat  Detection:
intake of 100 g/d before and during collection  Patients should include meat in their diet prior to
– Specimen are refrigerated; specimen is weighed collection
and homogenized prior to testing  Emulsified stool + 2 drops 10% eosin in alcohol 
– Methods: normal 1-6 g/d; steatorrhea: >6 g/d coverslip, stand for 3min  undigested muscle
1. Van de Kamer titration (only detects 80% of fibers counted using HPO within 5min
total fat contents)  Creatorrhea: >10 undigested muscle fibers
 Gold standard for fecal fat determination
 Titration using sodium hydroxide

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TESTS FOR FECAL BLOOD  Does not detect blood from other sources such as
bleeding sources, therefore, lowering false (+)
Occult Blood Tests
 Detection of occult blood is the most frequently PROPHYRIN-BASED FECAL OCCULT BLOOD TEST (pFOBT)
performed test for fecal analysis  Principle: fluorometric test for hgb based on the
 Bleeding >2.5 mL/150 g of stool is considered conversion of heme to fluorescent porphyrins
significant  More sensitive to upper GI bleeding
 Annual testing for occult blood has a high (+)  False (–): same as gFOBT
predicative value for the detection of colorectal cancer  False (+): red meat/non-human sources of blood

GUIAC-BASED FECAL OCCULT BLOOD TEST (gFOBT) APT (apt-Downey) Test

 Patient to collect samples for three consecutive days  Distinguish fetal blood and maternal blood in an
 Principle: pseudoperoxidase activity of hgb infant’s stool or vomitus
 False (–): vitamin C (avoided 3d prior), iron therapy,  Emulsified stool  centrifuged  collect supernatant
failure to wait for specified time (2 tubes), 1 tube + 1% NaOH  compare color
 False (+):  Results:
 Turnips, broccoli, cauliflower, banana, apple,  Pink solution = hgb F
melon, horseradish, red meat (avoid for 3d)  Yellow-brown supernatant = maternal hgb
 Aspirin, aspilet (promote GIT bleeding; avoid for 7d)
DETECTION OF FECAL LEUKOCYTES
IMMUNOCHEMICAL FECAL OCCULT BLOOD TEST (iFOBT)  Fecal leukocytes are primarily neutrophils in the feces
 Specific for the globin portion of human hgb  70% sensitivity for invasive conditions are indicated
 Does not require dietary or drug restrictions (even by a count of 3 neutrophils per HPF
using aspirin and other anti-inflammatory drugs)  Diarrhea with WBCs: Salmonella, Shigella,
 More sensitive to lower GI bleeding, thus, a better Campylobacter, Yersinia, EIEC
indicator for colorectal cancer  Diarrhea without WBCs: parasites, viruses,
Staphylococcus aureus, Vibrio spp.
WET PREPARATION DRIED PREPARATION LACTOFERRIN LATEX AGGLUTINATION TEST
 Fresh specimen stained with Wright’s/Gram  Sensitive detection of fecal leukocytes even in
 Fresh specimen stained with
stain refrigerated and frozen specimens
methylene blue
 Additional advantage of Gram stain is the  The presence of lactoferrin (secondary
 Faster procedure than dry smears but
differentiation between Gram (+) and Gram granulocyte granules) is indicative of invasive
may be more difficult to interpret
(–) bacteria bacterial pathogen

FECAL CARBOHYDRATES  Normal stool pH: 7.0-8.0

 Most valuable in assessing cases of infant diarrhea – Stool pH in the presence of carbohydrate disorders :
(e.g. lactose intolerance) 5.5
 Most commonly used test: clinitest  Follow up tests: D-xylose (malabsorption) and lactose
– Result of >0.5 g/dL is indicative of carbohydrate tolerance test (maldigestion)
disorders

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