Professional Documents
Culture Documents
CHEMISTRY I
MKEB 2404
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
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pH Increased or decreased
Glucose decreased
Ketones decreased
Bilirubin decreased
Urobilinogen decreased
Nitrite Increased or decreased
Microscopic changes
Decreased due to disintegration especially in alkaline
urine
RBC, WBC, casts RBC decreased after 6 hours
WBC decreased 50% within 3 hours
Hyaline and granular casts decreased after 2 hours
Bacteria Increased due to bacterial proliferation
Precipitation of uric acid, calcium phosphate and
calcium oxalate
Yeast cells develop pseudo-mycelia
Others 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:
Physical examination
Color 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
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Amber – urobilin - no yellow foam
Dark amber – bilirubin - yellow foam
Dark amber – biliverdin - imparts green blue
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
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Riboflavin – bright - yellow
Clear, slightly cloudy, cloudy, turbid
Turbidity or cloudy urine:
Amorphous salts – non pathologic
Bacteria, blood cells - pathologic
Causes of turbidity (pathologic):
RBC, WBC
Bacteria, yeast, trichomonas
Renal epithelial cells
Clarity and
Fat (lipids, chy;e)
Odour
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
Dipstick – testing Well-mixed uncentrifuged urine sample
procedure Dip strip into urine briefly
(manual) Remove excess urine
Read colour development according to manufacturer’s
instruction
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Read in a well lit area
Aware of false positive and false negative results
Reaction:
Pseudoperoxidase action of Hgb myoglobin catalyzes
the oxidation of chromogens to produce a color change
False negatives:
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
Blood
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)
Bilirubin 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:
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:
Glucose 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.
β-hydroxybutyerate 78%, acetoacetate 20%,acetone 2%
False negative:
Delay in examination
False positives:
Ketones
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
Leukocytes Reaction :
Leukocyte esterase, present in granulocytes, catalyzes
the reaction of the chromogens to produce a color
change.
False negative:
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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
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.
Nitrites False negative:
Elevated SG, urobilinogen, pH <6.0, excess ascorbic
acid
False positives:
Presence of red dyes or other chromogens,
contamination
pH 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
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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 albumin
False positives:
Alkaline or concentrated urine, quaternary ammonia
compounds
Protein
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,
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purple.
False negative:
Excess nitrites; presence of formalin
False positives:
Presence phenazopyridine; very warm urine, elevated
nitrite levels
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Appear as pale discs
Can be confused with yeast cells
Red blood cells
Yeast cells do not stain and are not lysed by the
addition of acetic acid
Increased numbers in conjunction with RBC cast
Dysmorphic bleeding assumed to be renal in origin
RBCs Absence of casts and protein - bleeding assumed to be
non-renal
Increased numbers are seen:
o Renal diseases
Leukocytes o Urinary tract infection
When accompanied by casts:
o Renal in origin
Line the distal 1/3 of the urethra
Squamous
Large numbers in women maybe a source of
epithelial cells
contamination
Line the urinary tract from the renal pelvis to the
Transitional
proximal 2/3 of the urethra
epithelial cells
few are present in normal urine
Small numbers maybe seen in normal urine
o Sloughing of aging cells
Renal tubular
Increased numbers are seen:
epithelial cells
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
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
Hyaline cast Translucent with brightfield microscopy
Increased numbers:
o Pyelonephritis, chronic renal disease
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o Transiently with exercise
o May be a normal finding
Associated with tubular inflammation and
Waxy cast degeneration
Observed frequently with chronic renal failure
Appear with glomerular and tubular diseases
Accompany:
Granular casts o Pyelonephritis
o Viral infections
o Chronic lead poisoning
Commonly seen when there is heavy proteinuria
Fatty casts A feature of nephrotic syndrome
Hypothyroidism
Diagnostic of glomerular disease
Red blood cells
Glomerular damage allows RBCs to escape into the
casts
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
Crystals found in Amorphous urates / phosphates
normal urine Calcium oxalates
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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:
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
Procedure for Low power microscopy:
microscopic Ensure uniform distribution of urine sediment
examination If uneven distribution, make a new preparation
Reduce light intensity
Scan whole area. Note: sediments tend to gather along
sides of cover-slip
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High power microscopy:
Examine 20 - 30 fields (optimal) but not less than 10
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
1 - 4 cells/HPF
5 - 9 cells/HPF
10 - 19 cells/HPF
20 - 29 cells/HPF
30 - 49 cells/HPF
50 - 99 cells/HPF
Numerous -100 cells and more
Casts:
- :0
Procedure for + : 1 cast/100LPF or 1 cast/WF
microscopic ++ : 1 cast/LPF or 100 casts/WF
examination +++ : 10 casts/LPF or 1,000 casts/WF
reporting format: ++++ : 100 casts/LPF or 10,000 casts/WF
or 6 casts/HPF
Bacteria and yeast:
- :0
+/- : scatter in several fields
+
++
3
: seen in each foeld
: many or scatter in cluster
+++ : numerous
Crystals and amorphous materials:
- :0
1 +
++
W : 1 ~ 4/HPF
: 5 ~ 9/HPF
W
+++: 10 ~ /HPF
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W W
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