DIAGNOSTIC IN CLINICAL
CHEMISTRY I
MKEB 2404
EXAMINATION OF BODY FLUIDS
Type of body fluids:
 Urine
 Seminal fluid
 Amniotic fluid
 Cerebrospinal fluid
 Synovial fluids
 Pleural, pericardial and peritoneal fluid
 Fecal analysis
First morning urine collection:
 Voids before going to bed
 Formed elements are more stable
 Unsuitable for cytology studies
 Collection of midstream urine for avoid contamination (let the first urine
throw first and only take the middle urine)
Urine container:
 Wide mouth (4 - 5 cm)
 Sufficient volume (50 ml preferred)
 Glass or plastic with no additives
 Leak-proof
 Sterile, if specimen is stored for a period of time before testing
The importance of urinalysis: as an indicator of health or disease, especially with
metabolic and renal disorders
Potential changes in unpreserved urine:
Physical changes
Bilirubin - biliverdin
Color
Hemoglobin - methemoglobin
Urobilinogen - urobilin
Clarity
Decreased due to bacterial proliferation,solute precipitation
Odour
Increased due to bacterial proliferation and decomposition
Chemical changes
pH
Increased or decreased
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Glucose
Ketones
Bilirubin
Urobilinogen
Nitrite
RBC, WBC, casts
Bacteria
Others
decreased
decreased
decreased
decreased
Increased or decreased
Microscopic changes
 Decreased due to disintegration especially in alkaline
urine
 RBC decreased after 6 hours
 WBC decreased 50% within 3 hours
 Hyaline and granular casts decreased after 2 hours
Increased due to bacterial proliferation
 Precipitation of uric acid, calcium phosphate and
calcium oxalate
 Yeast cells develop pseudo-mycelia
 Spermatozoa become immobile
 Trichomonas become immobile, maybe counted as
WBC
 Contamination by air borne particles
Urine examination:
 Most preservatives prevent bacterial growth and loss of glucose (eg.
Stabilur, formalin)
 No preservatives can prevent destruction of bilirubin, urobilinogen or
occult blood.
 Use of preservatives may increase SG, minor effects on pH and may
inhibit leukocyte esterase reaction.
 No single urine preservative is available
Urine FEME:
Color
Physical examination
 Urochrome, urobilin, uroerythrin
 Normal color range from straw, pale yellow, to amber.
Abnormal color:
 Red - RBCs
 Beer-brown - bilirubin
 Orange, blue, green - drug, dye or food
 Colorless - dilute urine - fluid ingestion: polyuria
 Light yellow and yellow - normal Urine
 Amber
- concentrated urine - dehydration, fever
 Amber – urobilin - no yellow foam
 Dark amber – bilirubin - yellow foam
 Dark amber – biliverdin - imparts green blue
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Orange – bilirubin - yellow foam if sufficient bilirubin
Orange – urobilin - no yellow foam
Orange – medication
Red - hemoglobin, red blood cell
Red – myoglobin - muscle injury
Red - porphyrins
Red – beets - genetic
Red - fuscin, analine dye - food, candy
Pink – hemoglobin, porphyrins
Brown – hemoglobin, myoglobin, methemoglobin
(muscle), homogentisic acid (acid pH), melanin
 Black – melanin (upon standing), homogentisic acid
(upon standing : alkaline urine)
 Green blue – indican (infections of small intestines),
chlorophyl (breath deodorizers), pseudomonas
infection, dyes and medication
Color changes due to oxidation:
 RBC oxidizers (brown) → methemoglobin (black)
 Urobilinogen (colorless) → urobilin (orange – brown)
 Porphobilinogen (colorless) → oxidizers to
porphobilin (red / purple)
 Bilirubin (amber) → oxdizers to biliverdin (greenish)
Urine color changes with commonly used drugs:
 Alcohol, ethyl – pale, diuresis
 Anthraquinone laxatives – reddish – alkalinr, yellow
brown – acid
 Chlorzoxazone (muscle relaxant) – red
 Deferoxamine mesylate (desferal) – red
 Furazolidone (an antibacterial, anti protozoal
pitrofuran) – brown
 Indigo carmine dye (renal function, cytoscopy) – blue
 Iron sorbital (jectofer) – brown on standing
 Leyodona (parkinsonism) – red then brown, alkaline
 Alcohol – pale
 Desferal and paraflex (muscle relaxant) – red
 L – Dopa (parkinsonism) – red then brown
 Flagyl – reddish brown
 Nitrofurantoin – brown – yellow
 Riboflavin – bright - yellow
Clear, slightly cloudy, cloudy, turbid
Turbidity or cloudy urine:
 Amorphous salts – non pathologic
 Bacteria, blood cells - pathologic
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Clarity and
Odour
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Causes of turbidity (pathologic):
 RBC, WBC
 Bacteria, yeast, trichomonas
 Renal epithelial cells
 Fat (lipids, chy;e)
 Abnormal crystals, calculi and pus
Causes of turbidity non pathologic:
 Normal crystals like urates and phosphates
 Radiographic media
 Mucus, mucin, squamous epithelial cells
 Sperm, posthatic fluid
 Salves, lotions, cream
 Powders, talc
Chemical examination
 Volume - average of 1.0 to 1.5L of urine excreted per
day
 Amount excreted is an indicator for diuretic disorder
Urinalysis
 Polyuria: More than 2000ml urine/day
 Oliguria: Less than 500ml urine/day
 Anuria : Less than 200ml urine/day
 Dysuria: No urinary excretion
 Example is Bayer – ames multistix
 Manual – subjective
Dipstick
 Machine – standardized reflectance photometer that
methods
measures scattered or reflected light, have multiple
channels and compensator pad
 Store in original container
Dipstick
 Do not expose to light, heat and moisture
methods – care
 If there is any colour change, discard
and storage
 Do not use pass expiration date.
 Store at manufacturer recommended temperatures
 Well-mixed uncentrifuged urine sample
 Dip strip into urine briefly
Dipstick – testing
 Remove excess urine
procedure
 Read colour development according to manufacturer’s
(manual)
instruction
 Read in a well lit area
 Aware of false positive and false negative results
Reaction:
 Pseudoperoxidase action of Hgb myoglobin catalyzes
Blood
the oxidation of chromogens to produce a color
change
False negatives:
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Formalin, excess nitrites (2.2 mmol/l), elevated SG,
ph <5.1, captopril, ascorbic acid
False positive:
 Oxidizing detergents, microbial peroxidase (UTI),
dehydration, exercise, hemoglobinuria,
myoglobinuria, menstrual contaminants, proteinuria
(5 g/l)
Hematuria:
 Presence of an abnormal number of blood cells in
urine as microhematuria or gross hematuria (0.5ml or
2500 RBC/µl)
 Occurs with disease or trauma anywhere in the
kidneys or urinary tract
 Can be seen in healthy persons undertaking excessive
exercise (marathon runners) in whom bleeding
emanates from the bladder mucosa. Repeat urinalysis
after 48 – 72 hours should be negative
 Causes: cancer, trauma, stones, infections,
obstructions, viral infections, inflammation of kidneys,
benign prostate enlargement, and warfarin therapy
 Calculi – ca oxalate (60%), uric acid (25%) phosphate
(20%)
 Tumors – painless hematuria
 Glomerulonephritis – hematuria with proteinuria
 Urinary tract infection
Separate sales:
 Green dots (intact RBC)
 Homogenous green color scale (for lysed RBC)
Reaction:
 Bilirubin in the urine couples with a diazonium salt in
an acid medium
False negative:
 Samples exposed to light, excess levels of ascorbic
acid.and nitrite, selenium, chlorpromazine
False positives:
 Highly colored metabolites of drugs eg pyridium
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 Breakdown product of hemoglobin formed in the in
the RES, liver, and bone marrow
 carried in the blood by protein
 Normal adult urine contains 1 mg/dL and this is not
detected by usual tests.
Reaction:

Bilirubin
Glucose
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Double sequential enzyme reaction of glucose oxidase
and peroxidase-reacts with a chromogen to produce
the final color.
False negative:
 Elevated specific gravity, uric acid, ascorbic acid
False positives:
 presence of oxidizing agents, ketones, levodopa
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 May appear in the urine and is influenced by:
o Blood glucose levels
o Glomerular blood flow
o Tubular reabsorption rate
 Often regarded as a hallmark of disease and requires a
patient to receive a workup for diabetes mellitus.
Reaction (Legal or rpthera’s test):
 Reaction with nitroprusside or sodium
nitroferricyanide and glycine to produce a color
change.
 -hydroxybutyerate 78%, acetoacetate 20%,acetone 2%
False negative:
 Delay in examination
False positives:
 Highly pigmented urines; some drug metabolites,
acidic urine, elevated SG
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 Products of incomplete fat metabolism
 Presence is indicative of acidosis
 Low carbohydrate diet for weight reduction will
produce ketonuria
 Exposure to cold and severe exercise
Reaction :
 Leukocyte esterase, present in granulocytes, catalyzes
the reaction of the chromogens to produce a color
change.
False negative:
 Cephalexin and gentamicin concentrations; elevated
SG, glucose, ketone and protein concentrations,
ascobic acid
False positives:
 Vaginal contaminants, drugs or foods that color the
urine red
Reaction :
 Nitrates in the urine are converted to nitrites by the
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Ketones
Leukocytes
Nitrites
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pH
Protein
action of gram-negative bacteria. These nitrites then
react to form a diazonium salt which in turn reacts
with a chromogen to produce the final color.
False negative:
 Elevated SG, urobilinogen, pH <6.0, excess ascorbic
acid
False positives:
 Presence of red dyes or other chromogens,
contamination
Reaction:
 Double indicator system detects the amount of
hydrogen ions in the urine to produce a color change.
Interferences:
 If excess urine is left on the reagent strip, a
phenomenon known as “runover” may occur. The
urine from one reagent area carries reagent onto the
pH test area and changes the result erroneously.
 Reflection of the ability of the kidney to maintain
normal hydrogen ion concentration in plasma and
extracellular fluid
 Normal adult: 4.6 - 8.0 pH
o Hypertonic urine < 6.0 - crenated RBC
o Hypotonic urine > 7.5 - Lysis of cells
 Acid urine: diet high in meat protein
 Alkaline urine: diet high in citrate or vegetables
 RTA type I (renal tubular acidosis)
o Serum is acidic, urine is alkaline
 RTA type II
o Urine initailly alkaline but becomes more acidic
due to decrease in bicarbonate load
 Useful in diagnosis and management of UTI and
calculi
o Alkaline urine in UTI suggests presence of
urea-splitting organisms
o Magnesium-ammonium phosphate crystals can
form staghorn calculi
o Uric acid calculi associated with acidic urine
Reaction :
 Based on “protein error of indicators” - because
protein carries a charge at physiologic pH, their
presence will elicit a pH change
False negative:
 Acidic or diluted urine, primary protein is not
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albumin
False positives:
 Alkaline or concentrated urine, quaternary ammonia
compounds
 High levels in urine indicates renal disease:
o Glomerular disease
o Tubular disease
 Functional proteinuria
o After strenuous exercise
Other methods of detection:
 Heat
 Acid (SSA- sulfosalicylic acid precipitation test)
Sensitivity: 5-10 mg/dL of protein
Reaction : ionic specific gravity
 Based on the change of an indicator color in the
presence of high concentrations of various ions.
False negative:
 Highly alkaline urine
False positives:
 Proteinuria, Dextran solutions,IV radiopaque dyes,
Specific gravity
Urobilinogen
 Random SG - 1.015 - 1.025
 SG< 1.010 indicates relative hydration
 SG > 1.020 indicates relative dehydration
 SG - 1.000 should be checked
 SG - 1.040 physiologically impossible
 Other methods: urinometer and refractometer
 Correlates with urine osmolality
 Insight to patient’s hydration status
 Reflects concentration ability of the kidneys
Reaction : ionic specific gravity
 Urobilinogen reacts with a chromogen to form an azo
dye which appears as various shades of pink or
purple.
False negative:
 Excess nitrites; presence of formalin
False positives:
 Presence phenazopyridine; very warm urine, elevated
nitrite levels
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Elevated urobilinogen found in hemolysis and
hepatocellular diseases
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Test strip sieve
technique
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Sediment
examination
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Microscopic
components in
urine sediment
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Methods for
examining urine
sediment
Red blood cells
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Dysmorphic
RBCs
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Leukocytes
Decrese robilinogen levels can be due to antibiotic use
and bile duct obstruction
Leucocyte
RBC
Protein
Nitrite
pH  7.0
Microscopic examination
Most common laboratory procedure utilized for the
detection of renal and/or urinary tract disease
Numerous morphologic entities
o Blood cells, epithelial cells, organisms
Correlate with the biochemical results
o Dipsticks
o Clinical condition of the patient
Cells
o Blood cells; RBCs and WBCs
o Epithelial cells; renal, transitional, squamous
Casts
o Hyaline
o Waxy
o Inclusion casts; Granular, Fatty
o Cellular; RBC, WBC, and Epithelial casts
Bacteria, Fungi, and Parasites
Crystals
Bright field Microscopy of unstained urine and w/
Supravital staining
Phase Contrast Microscopy
Polarized Microscopy
Interference Contrast Microscopy
Cytodiagnostic Urinalysis
Quantitative and Differential Counts
Appear as pale discs
Can be confused with yeast cells
Yeast cells do not stain and are not lysed by the
addition of acetic acid
Increased numbers in conjunction with RBC cast
bleeding assumed to be renal in origin
Absence of casts and protein - bleeding assumed to be
non-renal
Increased numbers are seen:
o Renal diseases
o Urinary tract infection
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Squamous
epithelial cells
Transitional
epithelial cells
Renal tubular
epithelial cells
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Casts
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Hyaline cast
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Waxy cast
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Granular casts
Fatty casts
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When accompanied by casts:
o Renal in origin
Line the distal 1/3 of the urethra
Large numbers in women maybe a source of
contamination
Line the urinary tract from the renal pelvis to the
proximal 2/3 of the urethra
few are present in normal urine
Small numbers maybe seen in normal urine
o Sloughing of aging cells
Increased numbers are seen:
o Acute tubular necrosis
o Certain drug or heavy metal toxicity
Formed when an increased numbers of proteins enter
the tubules.
Formation increases with:
o Lower pH
o Increased ionic concentration
Tamm-Horsfall (TH) protein forms the matrix of all
casts
o Glycoprotein secreted by cells in the ascending
loop of Henle
If cast contain 3 or more cells e.g. RBC, WBC, then it is
RBC cast, WBC cast
If it contains 1/3 or more granules - granular cast
If a cast is about 60 µm or more - broad cast, RBC 7-8
µm, WBC 8-22 µm
Translucent with brightfield microscopy
Increased numbers:
o Pyelonephritis, chronic renal disease
o Transiently with exercise
o May be a normal finding
Associated with tubular inflammation and
degeneration
Observed frequently with chronic renal failure
Appear with glomerular and tubular diseases
Accompany:
o Pyelonephritis
o Viral infections
o Chronic lead poisoning
Commonly seen when there is heavy proteinuria
A feature of nephrotic syndrome
Hypothyroidism
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Diagnostic of glomerular disease
Glomerular damage allows RBCs to escape into the
tubules
 WBCs enter the tubular lumen through and between
White blood cells
tubular epithelial cells
casts
 Associated with pyelonephritis and tubulointerstitial
disease
 To differentiate from leukocyte cast, supravital
Epithelial cells
staining and phase -contrast microscopy are helpful.
casts
 Associated with: tubular necrosis, viral disease (CMV),
heavy metal ingestion
Other component that also found is bacteria, fungi, and parasites and also clue
cells
 Limited clinical significance
 Phosphates, urates, and oxalates are common and
occur in normal urine
 Alkalization and refrigeration promotes crystals
formation
 Few crystals are important:
Crystals
o Cystine
o Tyrosine
o Leucine
o Bilirubin - hepatic and biliary tract diseases
o Cholesterol - nephrotic syndrome
 Cysteine, leucine and tyrosine is due to inherited
metabolic disorders
 Amorphous urates / phosphates
Crystals found in
 Calcium oxalates
normal urine
 Uric acid
 Triple phosphates
 Cysteine
Crystals found in
 Bilirubin
abnormal urine
 Tyrosine
 Cholesterol
Procedure for
Sample collection → centrifugation → decantation → slide
urine microscopy preparation → microscopy → writing report
Centrifuge or not to centrifuge:
“After a 5 min centrifugation of the urine at 3500rpm, only 48%
Quantitative ME
of RBC and 40% of WBC found to be present could still be detected
(microscopic
under the microscope”
examination)
To report in µl or LPF or HPF
To stain or not to stain
Neubauer
See picture below:
Red blood cells
casts
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counting
chamber
Analysis volume of Neubauer is half of Fuch
Rosenthal
o Concentration of urine formed elements is very
Fuch Rosenthal
low compared with those of hematology.
vs Neubauer for
o For urinalysis, the more volume, the better
quantitatve urine
results
microscopy:
 Some urine formed elements are large (ie casts), these
Advantages of
might clog the chamber
Fuch Rosenthal
o The deeper the depth, the better
over Neubauser:
 Cells /uL = total cells counted / [mm2 counted (how
many mm squares were conted) x 0.1 mm (neubauer
chamber) or 0.2mm (fuch rosenthal chamber)]
Depends on:
 Real View ( Diameter )
 Magnification# ( x10 or x40 )
HPF LPF or µl
 Original Urine Volume before Centrifugation
conversion
 Sediment Volume after Centrifugation
 Loaded Sediment Volume on the Slide
 Area of Cover Slip
Low power microscopy:
 Ensure uniform distribution of urine sediment
 If uneven distribution, make a new preparation
 Reduce light intensity
 Scan whole area. Note: sediments tend to gather along
sides of cover-slip
Procedure for
High power microscopy:
microscopic
 Examine 20 - 30 fields (optimal) but not less than 10
examination
fields
Microscopic with staining sediment to stain 4:1
Types of stain:
1. Sternheimer-Malbin Stain (SM Stain)
2. Sterheimer Stain (S Stain)
3. 0.5% Toluidine Blue
4. Sudan III and Oil Red O
Blood cells:
 Less than 1cell/HPF
Procedure for
 1 - 4 cells/HPF
microscopic
 5 - 9 cells/HPF
examination
 10 - 19 cells/HPF
reporting format:
 20 - 29 cells/HPF
 30 - 49 cells/HPF
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Casts:
+
++
+++
++++
50 - 99 cells/HPF
Numerous -100 cells and more
:0
: 1 cast/100LPF
: 1 cast/LPF
: 10 casts/LPF
: 100 casts/LPF
or 1 cast/WF
or 100 casts/WF
or 1,000 casts/WF
or 10,000 casts/WF
or 6 casts/HPF
Bacteria and yeast:
:0
+/- : scatter in several fields
+
: seen in each foeld
++
: many or scatter in cluster
+++ : numerous
Crystals and amorphous materials:
:0
+
: 1 ~ 4/HPF
++
: 5 ~ 9/HPF
+++: 10 ~ /HPF
3
mm
1
mm
W
W
W
W
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