Physical, chemical and

microscopic urine
examination

Violeta BLAGA
Definition
 Urine = a product of glomerular filtration,
tubular reabsorption and secretion

Chemical composition
 Water: 93 - 95%
 Mineral and organic substances: 7-5%
 Organic substance 60% (urea, uric acid,
creatinin, proteins, glucoses, amino acids,
ketones, urobilinogen, hormones etc.)
 Mineral substance 40% (chlorides, sulphates,
phosphates, Na, K, ammonium salts, Ca, Mg)
Urine sample examined:
 First fresh morning urine (glucose, albumin, pH,
urobilinogen, bilirubin, ketones and urine sediment)
 Spot urine
 Special collection of urine (e.g. for Addis method)
 24 h urine: after the urinary bladder was emptied of
the “morning urine”, the collection begins at a precise
time (e.g. from 7:00 this morning until tomorrow
morning at the same hour)
Urine collection
Urine collection is to be avoided in:
 Women during the menstrual cycles and 2-3
days after
 Patients radiologically investigated using
contrast media in the previous 48 h (density >
1040 and false+ reaction at proteins)
 Cease of the aspirin or sulphonamide
medication (increased erythrocyte flow) and
also the cease of the diuretics and laxatives.
 Water restriction 12 h before. Densities <1015
impose to repeat the exam.
Urine collection

If urine is examined more than 3 hs after

emission, are expected:
 Red blood cells destruction
 Leukocytes degradation
 Fast bacterial growth
 Appearance of nitrites
 pH increase (the urea is transformed in
ammonia under the bacteria influence)
 pH increase can produce cast dissolution
Urine samples conservation

 Can be done with:

 Thymol
 Methyl-4-hydroxibenzoate
 Chlorophorm
 Formol
 Phenol
 Toluene
 Conservation at +4º C
Quantity

 Normal diuresis 1000-1800 ml/day

 OLIGURIA (volume < 800 ml/day)

 POLYURIA (volume > 2000 ml/day)
 ANURIA (volume < 300 ml/day)
Macroscopic urinalysis

 Aspect
 Colour
 Smell
Macroscopic urinalysis

The aspect
 Fresh, normal urine must be clear
 Fresh, turbid urine may contain:
 Salts (urates, uric acid, oxalates,
phosphates or carbonates)
 Mucus, epithelia, microorganisms
 Lipids (milky aspect)
Macroscopic urinalysis
Heating test (5-6 ml urine in a test tube)
 Turbidity disappears: urates , uric acid
 Turbidity increases: proteins, carbonates or
phosphates
 Turbidity does not disappear, but clears up after:
 Adding some drops of acetic acid 10%:
phosphates, carbonates (with CO2 release)
 Adding 2-3 ml of HCl 12,5%: oxalates,
leucine, tyrosine, cystine
 Adding 2-3 ml of NaOH 20%: uric acid,
mucus, cystine
 Adding a mix of ethanol – ether: lipids
Macroscopic urinalysis

 Aspect
 Colour
 Smell
Macroscopic urinalysis
Colour
 The urine normal colour is from light
yellow to gold yellow and it is
determinate by pigments: urochrome,
urobiline, uroerithrine.
 Macroscopic urinalysis
Colour
 The abnormal urine colour can be induced by many exogenous
and endogenous substances
 Milky white:
 chyluria
 From yellow to uncoloured:
 Watery diuresis, diabetes insipidus, diuretic treatment
 From yellow saffron to brown:
 carotenes, flavones, quinine, phenolphtaleine,
bilirubin, erythrocytes, haemoglobin, myoglobin,
porphobilin,
 Yellow orange:
 Low fluid intake, fever, sulphonamides, urobilinogen
Macroscopic urinalysis
Colour

 Red orange
 betanidine, alimentary colorants,phenitoin,
aminophenazone, nitrofurantoin, metronidazole,
pirogalol, haemoglobin, uroglobin, erythrocytes,
porphyrines
 Red
 Fresh blood, red beet, haemoglobin, myoglobin
 Dark red
 porphyrin
Macroscopic urinalysis
Colour
 Brown
 tanin, thymol, indican, porphobilin
 Green blue
 indigo-carmine, methylene blue, biliverdin,
copper
 Dirty green
 indigo-carmine, acriflavine, methylene blue,
copper, biliverdina, indican, chromogen bacteria
(Pseudomonas spp - piocianic)
Macroscopic urinalysis

 Aspect
 Colour
 Smell
 Macroscopic urinalysis
Smell
 Normal fresh urine has a specific smell because of the
volatile acids
 concentrate urine has a powerful smell
 diabetic urine – sour apples smell

 alcoholic urine - alcohol

 infected urine or renal tumours - ammonia

 in acidosis - fruits or chloroform smell

 drugs based on turpentine essence - violet

 garlic, horse radish, asparagus – unpleasant smell

 new borns urine with powerful smell indicates a
metabolically error (aminoacidopathy)
Physical urine exam

 Density
 Osmolality
 Urine pH
Density
Depends on the dissolved substances concentration: salts
in general and urea at healthy people, glucose and
albumin in pathological cases
 Normal values: 1015-1022 with extremes: 1003- 1035
 Urodensimeter for 20º C
Density correction
 For every 3º C ± from the standard temperature we add
or decrease with one unit
 When urine contains glucose, proteins or crystals we
make a quantitative determination of their concentration
and reduce with one unit from the predetermined density
for each one: 2,5g glucose/l, 3.3 g proteins/l, 2.2 g
crystals/l.
Physical urine exam

 Density
 Osmolality
 Urinary pH
Osmolality

 Refers to the number of dissolved molecules in 1

kg solvent
 Osmometers evaluate osmolality by determining
urine freezing point, starting from the relation
0, 56ºC for 272 mOsm/kg. Measuring unit is
miliosmoles/kg
 Normal values: 800-1200mOsm/kg
 Osmolality is a measure of concentration capacity
of kidney.
Physical urine exam

 Density
 Osmolality
 Urinary pH
 Urines reaction – urinary pH

 Allows the evaluation of the kidneys intervention in

maintaining the acid-base equilibrium
 In normal individuals on a mixed diet, urine is usually
low acid (pH~6).
 Urine acidity comes from: organic acids, uric acid,
citric acid, acetic acid and also from acid salts –
sodium primary phosphates, K, ammonium.
 A hyper proteic diet produces an increase in urine
acidity by enhancing uric acid, urates and acid
phosphates elimination.
Urines reaction – urinary pH
 A vegetarian diet induces an alkaline urine
because of the excess of mineral and organic salts.
 Strongly acid urines (4-4.5 pH) - malignancy, fever,
diarrhoea, diabetic or metabolic acidosis
 Strongly alkaline urine – urinary infections,
respiratory alkalosis, metabolic alkalosis
 Urinary pH is determined with pH paper or dipstick.
Chemical urine exam

 Is done in order to evaluate different

urine components: proteins, glucids,
ketonic bodies, blood pigments,
urobilinogen, bile pigments, bile acids
Proteins

 Are measured in the 24 h urine collection

 Normal values
<150mg/24 h in adults
<140mg/m2 body surface in children

 Determination methods:
 Semi quantitative
 Quantitative
 Qualitative
Semi quantitative methods

a) Sulphosalicilic acid 20% method:

different types of proteins precipitate
(albumins, globulins, peptide, Bence –
Jones proteins, Tamm-Horsfall
lipoproteins, and albumoses – produced
by proteins degradation). Determined
turbidity is proportional with urine
proteins concentration
 Result Approximative Aspect
concentration of
proteins (mg/dL)
0 absent < 10 Clear

± very fine 10 - 15 Very weak turbidity

traces
+ fine traces 15 - 20 Well- defined turbidity

++ fine cloud 20 – 30 White cloud, precipitate (-)

+++ thick cloud 30 - 50 White cloud, precipitate (+)

++++ dosable >50 Flocculent precipitate

Semi quantitative methods

b) Warm coagulation method Warming a

urine sample at a temperature > 60º C
determines the proteins coagulation in
white flocculus which modify the sample
transparency according with the proteins
concentration
c) Dipstick method. The test specificity is
higher for albumins and lower for Bence-
Jones proteins
Quantitative methods

 Quantitative urinary proteins are determined:

 From 24 h urine samples (reported as g/24 h).
Most used determination methods are:
- 3% sulphosalicilic acid,
- pirogalol or Folin-Lowry method.
 Determination in spot urine:
- urinary proteins,
- urinary creatinine
Their ratio Upr/Ucr approximates 24hrs proteinuria.
Qualitative methods

 Offer data on physical – chemical

configuration and on urinary proteins
structure.
 Allow the classification of proteinuria
according to the determining process.
 Methods: electrophoresis , immune
electrophoresis, ELISA, immune radial
diffusion
Qualitative methods

 From the electrophoresis point of view

we can identify 3 types of proteinuria:
physiological, glomerular or tubular.
 Physiological proteinuria is the result of 3
big processes:
 glomerular filtration
 tubular reabsorption of filtrated proteins
 protein tubular secretion
Qualitative methods

 Pathological proteinuria results from 4

mechanisms:
 The increase in the glomerular filter permeability
 The decrease in the tubular reabsorption of
normally filtered proteins
 Modifications in the physical-chemical proteins
configuration (sudden increase of plasmatic
proteins concentration)
 Increase in tubular secretion which appears
through nephron degradation or excess tubular
proteins production.
 Bence-Jones proteins

 Contain proteins with MW 22000-45000 and sometimes

90000. The concentration of these proteins in serum is
<10µg/ml, and in pathological conditions can reach
100µg/ml. The concentration of these proteins in
pathological conditions is 1-6 g in 24 h.
 Diagnostic methods for Bence-Jones proteinuria:
 Precipitation at 56°C and redissolution at100°C
 Radial immune diffusion
 Immune fixation
 Bence-Jones proteinuria – most often encountered in
myeloma, but can also appear in leukaemia,
osteosarcoma and very rare in Waldenstrom
macroglobulinemia
 β2 - microglobulin

 Is a globular protein, MW=11800

 Can be found in serum, saliva, synovial liquid,
urine
 Normal values in urine: 4 - 370μg/l
 Increased values appear in: multiple myeloma
, chronic leukaemia, rheumatoid artritis,
sarcoidosis, lupus, hepatitis, hepatic cirrhosis,
immune deficiency syndrome
 Glucose
 Semi quantitative methods
Fehling
 Benedict
 Dipstick
 Quantitative methods
 Colorimetrical with orthotoluidine
 Enzymatic with hexokinase
 Glycosuria depends on:
 Glucoses concentration in blood
 Glomerular filtration rate
 Tubular reabsorption rate of glucose
Glycosuria is seen in: diabetes mellitus, renal diabetes, renal
hereditary glicozuria, Fanconi syndrome, stress, acidosis, CNS
lesions, 10-15% of pregnant women.
 Urobilinogen

 Is a halogenated derivate of bilirubin; together with urobilin,

stercobilinogen and stercobilin – normal values 4mg/24 h.
 Increased excretion indicates diseases:
 With excess production: hemolytic anaemia, macrocytic
anaemia, policitemia vera. Enterocolitis.
 True functional hepatic deficiency: hepatitis ac. and cr., cirrhosis.

 The urobilinogen is absent if the coledoc channel is completely

obstructed.
 The urobilinogen determination in urine is done through Erlich
classic method or with dipstick. We add 5- 10 ml Erlich solution
drops in 5 ml urine:
 Red colour at room temperature - urobilinogen ;

 Red colour after warming – normal urobilinogen

 If the red colour doesn’t appear after warming – absent

urobilinogen
Microscopic urinalysis
URINARY SEDIMENT
 Preparing the urine sample:
 We agitate the recipient with the sample from the
first morning urine
 We put it in a conic test tube of minimum 10 ml
 We centrifuge it 2-3 minutes at 1000 rpm
 We keep the supernatant for protein determinations.
 From the sediment we use 0.5-1ml to prepare the
sample for analyzing the urinary sediment
 It is well agitated and after, a drop is put on to the
thin plate and than it’s covered with a lamella.
 The examination is done with 20X and 40 X
objectives.
Microscopic examination methods
 Optic microscopy – the most used in clinical
laboratories
 Phase contrast microscopy is more sensitive
and gives an accurate erythrocyte and cast
evaluation
 Interface microscopy – offers a three-
dimensional image
 Polarized light microscopy – detects the bi
refringent corps and the crystals.
 Immune fluorescent microscopy: allowed the
cast matrix identification
 Electronic microscopy
Examination techniques

 The examination in fresh drop is the most used

 Coloured sediment examination – brings more
information in the appreciation of the structure;
methods:
 Sternheimer-Malbin – casts of leucocytes

 Wright – dysmorphic erythrocytes

 Giemsa

 Hansen - eosinophiluria

 Papanicolau method for cytological exam and

neoplastic cell detection
 Urinary sediment examination

 organized structures  unorganized structures

(metabolic origin)
 Epithelial cells
 Organic substances
 White blood cells  Uric acid
 Red blood cells  Cystine
 Casts  Leucine
 Bacteria  Tyrosine
 Inorganic substances
 Carbonates
 Oxalates
 Phosphates
 Semi quantitative microscopic
urine examination
A. Organized sediment:
1. Epithelial cells
 pavimentous epithelial
cells :
 big, flat cells with picnotic
nucleus and variable diameter
 come from the superficial
layers of the inferior urinary
tract
 Semi quantitative microscopic
urine examination
 transitional epithelial cells:
 spindle shaped or round
 visible nucleus
 come from desquamated
renal calices until the
proximal urethra segment
 are frequent in neoplasia or
inflammatory processes
 Semi quantitative microscopic
urine examination
 renal tubular epithelial cells:
 cubic or cylindrical shape
 smoothly granulated
cytoplasm and big, vesicular
nucleus
 marker for tubular lesions
 appear in infections, after
chemotherapy
 can be lipid-laden
 Semi quantitative microscopic
urine examination
 2. Leucocytes
 can come from the glomeruli until the
proximal urethra
 are mainly neutrofils

 in pyuria they are degenerated and

covered in mucus
 eosinophiluria appears in acute
interstitial allergic nephritis
 Leukocyturia: caused by infection,
inflammation, haemorrhage
 when there is no infection we can
suspect calculi, papillary necrosis or
renal polycystic kidney disease
 normal values: 1-2/field ; <10/mm³
(Stansfeld-Webb)
 Semi quantitative microscopic urine
examination
3. Erythrocytes (can come from the urinary
tract or woman genital tract)
 In normal sediment: 1-3 on microscopic
fields or 5/mm³
 Isomorphic erythrocytes:
 yellow biconcave disks with defined
contour and equal diameters
 Influenced by urine osmolality:
 isotonic urines: yellow, biconcave disks
 hypotonic urines: spherical shape
 hypertonic urines: may be crenelated
 Dysmorphic erythrocytes
 uneven colour, abnormal shape and
smaller, unequal diameters
 Haematuria
Macro haematuria
 Appears when there is more
than 0,5ml blood/1l of urine
(>2500 erythrocytes /μL)
 urine is coloured from red to
brown
Micro haematuria
 red blood cells
number<2500/μL
 urine colour stays the same
Glomerular hematuria
 accompanied by
erythrocyte casts, marked
proteinuria, dysmorphic
erythrocytes
Inferior urinary tract
hematuria
 isomorphic red blood cells
Red blood cells of glomerular origin

 Intact or fragmented
 Mainly decolorized (>80%)
 Appear as double rings, with smaller
diameter, are unequal and dysmorphic -
“phantom cells”
 Acanthocytes are predictive for
glomerular hematuria
Dysmorphic red blood cells
Casts

 The most important element in the urinary sediment for

establishing the differential diagnostic
 Are renal tubules mouldings mainly formed of Tamm-
Horsfall glycoprotein
 Cylindrical shape, round/smooth endings, but never
sharp
 Are formed by proteins jellification when proteins are in
excess in the tubular fluid, either by increased local
production or by increased ultrafiltration;
 The process is stimulated by:
 Urine concentration,
 Urine acidification - low pH 4,7-7 (in pH >7 it dissolves)
 Hyaline casts

 Matrix formed exclusively from

glycoproteins, are transparent,
uncoloured, variable length and
constant diameter
 Accompany proteinuria and appear in
large number in nephropathies
associated with oliguria, acid pH and
high proteinuria (nephrotic syndrome)
 Epithelial casts

 Have renal tubular

epithelial cells entraped
in their matrix and can
be loaded with oval fat
bodies
 Can appear in
association with
epithelial tubular free
cells and granular casts
 Are nonspecific markers
for tubular lesions
 Red blood cell casts

 Are identified after the

presence of isomorphic or
dysmorphic red cells on the
matrix surface
 Appear in glomerulonephritis,
renal transplant rejection
and, sometimes, in tubulo –
interstitial nephropathies
 Occasionally, can be
observed even in renal
trauma, cortical necrosis
 Markers of glomerular
haematuria .
 White blood cell casts

 Contain leukocytes with

different degrees of
degeneration within the protein
matrix
 Markers for acute or chronic
renal parenchymal infections;
their presence asks for careful
bacteriological investigations
 Appear in pyelonephritis and
sometimes in glomerulo-
nephritis, lupus nephropathies
or in aseptic kidney transplant
inflammation
 Granular casts
 Are formed through a progressive
degenerative process resulting in
granulations with different size
 Can have bigger or smaller
granulations
 Are bigger than hyaline casts, have a
net shape and round or broken
extremities
 The main characteristic of
granulations is their refringence
 Appear constantly in chronic
glomerular or interstitial
nephropathies and in acute tubular
necrosis
 Waxy casts

 Well-defined shape, mate

aspect, yellow pale
 Very fragile, with notched
margins and variable
dimensions
 Increased refraction index and
formed from clear and
homogeneous material
 Results from the granular cast
degeneration; these casts were
fixed in the distal tubules’
lumena for a long time
Other cast types
Fat casts
 Contain lipid globules covering the hyaline matrix surface; in
polarized light appear as Maltese cross.
 Appear in abundant proteinuria (nephrotic syndrome)
Cylindroids
 Ribbon shaped, with longitudinal striations and sharp
ending; usually one head is split
 Are formed from mucous/amorphous substances, probably
in the pyelon or urether, not in tubules
Pseudo casts
 Appear when urate or phosphate agglomerations are
produced accidentally in the renal tubules
 Can form shapes similar with casts
 Other pseudo casts can be formed from fibrin or pus
Lipid bodies
 Appear as free lipid drops and oval fat bodies
 Oval fat bodies result from excessive tubular
degeneration
Yeasts
 Ovalar or round, uneven dimensions, uncoloured and
can appear isolated or burgeoned
 Candida albicans appear under the microscope with
ovalar small cells shape, burgeoned or as a thin
filament (mycelium)
 Determinant factors yeasts collonization in urinary
tract favoured by: antibiotics, some hormones,
diabetes mellitus, avitaminosis
 Microorganisms
 The most frequent cause of
bacteriuria is urinary infection,
but can also appear by
contamination
 In infections, bacteriuria is
accompanied by leucocyturia
 Pathological circumstances
with negative urine cultures
and present leukocyturia:
urinary tuberculosis,
analgesic nephropathy,
mycotic infections and
perinephretic or cortical
abscess
 Unorganized sediment
Is represented by amorphous or crystalline salts of
metabolic, organic or inorganic origin

 a) Organic substances  Acid urine

 Calcium oxalate
 Uric acid
 Uric acid
 Cystine
 Amorphous urate
 Leucine  Alkaline urine
 Tyrosine  Ammonium urate
 Ammoniac- magnesium
 b) Inorganic substances
phosphate
 Carbonates  Calcium carbonate
 Oxalates  Calcium phosphate
 Amorphous phosphate
 Phosphates
 Quantitative urine microscopic
exam
 Quantitative microscopic exam can be done
through two methods:
 Addis-Hamburger method: after voiding, the patient
ingests 300mL of water, then rests in bed for 100
minutes, after that urine is collected. The results are
expressed as flow.
Normal values:
- Leucocytes <2000/minute
- Erythrocytes <1000/minute
 Stansfeld-Webb method
- Leucocytes <10/mm³
- Erythrocytes < 5/mm³