7. TIMING
ī Random sample- sufficient.
ī 1st specimen voided in morning is more
concentrated- preferred
ī 24 sample24 hours urine sample- quantitative
estimation of proteins, sugars, electrolytes, and
hormones.
ī 2-3 hours after eating- Glycosuria
ī Afternoon- For urobilinogen
11. Pathological
Nonpathological
Red to Brown to
Purple
Brown to Black
Porphobilinogen
Uroporphyrin
Homogenistic acid
Melanin
Myoglobin
Methaemoglobin
Phenol
Porphyrins
Blue to Green
Biliverdin
Acriflavine
Pseudomonas infection Azure A
Methylene blue
Vit B
Phenyl salicylate
Amitryptiline
Chloroquine
Iron compounds
Levodopa
Metronidazole
Quinine
12. APPEARANCE
ī NORMAL URINE- CLEAR
ī Cloudyī Precipitation of amorphous phosphates in alkaline urine
/ amorphous urates in acid urine.
ī Amorphous phosphates dissolve on addition of acetic
acid.
ī Amorphous urates will dissolve when specimen is
heated.
ī Turbid- Leucocytes , epithelial cells, bacteria
ī Hazy- Mucous
ī Smoky- RBC
ī Milky- Fat, Chyle
13. ODOUR
ī
Fruity/sweet odour- presence of ketones.
ī Pungent smell- presence of bacteria/ specimen
contaminated with bacteria.
ī Sweaty feet- Isolvaleric acidemia
ī Misty/mousy odour- Phenylketonuria.
ī Maple syrup- Congenital metabolic disorder.
ī Fishy odour/Rancid butter- Hypermethioninemia
14. pH
ī Concentration ability of kidney to maintain normal
hydrogen ion concentration
ī Normal pH â 4.6 to 8.0
ī Average- 6.0
ī PROCEDURE
ī Dip the litmus paper strips in the urine, remove and
read the color change immediately.
ī Blue litmus turns red â acid
ī Red litmus turns blue â alkaline
15. ī Decrease in pH
ī Increase in pH
ī High protein intake
ī Diet high in vegetables
ī Ingestion of
ī
ī
ī
ī
ī
ī
cranberries
Respiratory acidosis
Metabolic acidosis
Uremia
Severe diarrhoea
Starvation
UTI caused by E.coli
ī
ī
ī
ī
and citrus fruits
Respiratory alkalosis
Metabolic alkalosis
Vomiting
UTI caused by Proteus
and Pseudomonas
16. Specific gravity
ī Ratio of weight of a volume of urine to the
weight of the same volume of distilled water at a
constant temperature.
īMeasure the concentrating and diluting power of
kidney.
ī Concentrating ability of kidney is one of the
first function to be lost as a result of tubular
damage .
17. ī Specific gravity increases when fluid intake is low
and decreases when fluid intake is high.
ī Specific gravity varies throughout the day.
ī Range of 24 hour sample- 1.015- 1.025.
18. ī Hyposthenuria
ī Consistently low specific gravity, <1.007.
ī Due to concentration problem.
ī Hypersthenuria
ī Consistently high specific gravity
ī Due to deprivation of water.
ī Isosthenuria
ī Fixed specific gravity of 1.010
ī Indicates poor tubular reabsorption
20. Urinometer
ī It is a hydrometer that is calibrated to measure
the specific gravity of urine at a specific
temperature, usually at 200C.
ī Based on principle of buoyancy so the urinometer
will float higher in urine than in water, because
urine is denser.
ī Thus higher the specific gravity of a specimen,
the higher the urinometer will float.
21. ī Specific gravity is affected by presence of dense
molecules, protein and glucose.
ī Subtract 0.03 from specific gravity after
temperature correction for each 1 g/dl of protein
and 0.004 for each 1g/dl of glucose.
ī Temperature correctionī For every 30C below 200C, subtract 0.001 from the
reading and for every 30C above 200C, add 0.001.
22. Procedure
1.
2.
3.
4.
5.
6.
7.
8.
9.
Allow urine to reach room temperature.
Check urinometer periodically with distilled water to
see if its read 1.000.
Mix urine
Add to cylinder (approx 15 ml).
Remove any foam because bubbles interfere with the
reading of meniscus.
The hydrometer must not come in contact with the
bottom or the sides of the cylinder.
Allow it to float freely.
It is necessary to spin the urinometer so that it will float
in the center of the cylinder.
Read the bottom of the meniscus while looking at the
hydrometer at eye level.
27. Proteins in urine
ī Normal- upto 150 mg/24 hours or 10mg/100ml in
single sample.
ī Methodsī Heat and acetic acid test- The test is based on the
principle of heat coagulation and precipitation of
proteins by acetic acid.
ī Sulphosalicylic acid test- Sulphosalicylic acid
neutralizes protein cation, resulting in precipitation of
28. Causes of proteinuria
ī Pre-renal
ī Addisonâs disease
ī Fever
ī Eclampsia
ī Hypertension
ī Haemoglobinuria
ī Rhabdomyolysis
Renal
īAll cases of
glomerulonephriti
s
īNephrotic
syndrome
īPyelonephritis
âĸ Post renal
ī Lesions of renal pelvis, urethra (cystitis, prostatitis)
ī Severe UTI
29. Cause of Proteinuria as Related to
Quantity
DAILY PROTEIN EXCRETION
CAUSE
0.15 to 2.0 g
Mild glomerulopathies
Tubular proteinuria
Overflow proteinuria
2.0 to 4.0 g
Usually glomerular
> 4.0 g
Always glomerular
33. Classification of Proteinuria
PATHOPHYSIOLOGIC
FEATURES
CAUSE
Glomerular
Increased glomerular
capillary permeability
to protein
Primary or
secondary
glomerulopathy
Tubular
Decreased tubular
reabsorption of
proteins in glomerular
filtrate
Tubular or
interstitial disease
Overflow
Increased production
of low-molecularweight proteins
TYPE
34. Selected Causes of Proteinuria by Type
Glomerular
Primary glomerulonephropathy
īąMinimal change disease
īąIdiopathic membranous
glomerulonephritis
īąFocal segmental
glomerulonephritis
īąMembranoproliferative
glomerulonephritis
īąIgA nephropathy
35. Secondary glomerulonephropathy
īŧDiabetes mellitus
īŧCollagen vascular disorders (e.g.,
lupus nephritis)
īŧAmyloidosis
īŧPreeclampsia
īŧInfection (e.g., HIV, hepatitis B and
C, poststreptococcal illness, syphilis,
malaria and endocarditis)
īŧGastrointestinal and lung cancers
īŧLymphoma, chronic renal
transplant rejection
Glomerulonephropathy associated with the following drugs:
īHeroin
īNSAIDs
īGold components
īPenicillamine
īLithium
īHeavy metals
37. ī Selective proteinuria
ī When LMW proteins like albumin (MW- 66000) or
transferrin (MW-76000) are selectively excreted
through kidney.
ī Eg- all causes of nephrotic syndrome.
ī Non-selective proteinuria
ī When HMW protein like globulin, fibrinogen in
addition to LMW protein are excreted through
kidney
38. Proteins in urine
ī Heat and Acetic Acid Method
ī Procedure :
ī Take a long test tube and fill ž the tube with clear urine.
ī Boil the upper portion over a flame, the lower portion serves
as the control.
ī If proteins, phosphates or carbonates are present in the
urine a turbidity develops.
ī Add 1-3 drops of 10% glacial acetic acid.
ī Any turbidity due to phosphate precipitation will clear or if it
is due to carbonates they disappear with effervescence.
ī If it persists, it is due to albumin.
39.
40. Interpretation
ī Negative â No turbidity or cloudiness.
ī Trace â Cloudiness visible against a black
ī
ī
ī
ī
background ( 5 mg / dl).
1+ - Definite cloudiness without flocculation and
granularity
( 10 â 30 mg / dl ).
2+ - Heavy and granular cloudiness without
flocculation.
( 40 â 100 mg / dl).
3+ - Dense opaque cloud with marked flocculation
( 200 â 500 mg / dl) .
4+ - Thick cloudiness with precipitation
41.
42. Sulphosalicylic acid test
ī If urine is alkaline, it should be acidified.
ī Procedure:
ī 2ml of acidic urine taken in test tube.
ī Add an equal volume of 20% Sulphosalicylic
acid.
ī Mix thoroughly, allow it to stand for 10 minutes
and estimate the amount of turbidity.
ī Absence of cloudiness- Absence of protein.
ī If turbidity persists after boiling- Positive for
protein.
43. ī Negative : No cloudiness
ī Trace: Barely visible cloudiness.
ī 1+ : definite cloud without granular flocculation
ī 2+ : heavy and granular cloud without granular
flocculation
ī 3+ : dense cloud with marked flocculation.
ī 4+ : Cloudiness with precipitation
44. Quantitative estimation of protein
ī Esbachâs method using albuminometer.
ī Reagentsī Esbachâs reagent
ī Acetic acid
Picric acid
Citric acid
Water
ī pH paper
ī Instrument
ī Esbachâs albuminometer
45. ī Procedureī Fill Esbachâs albuminometer with
âĸ
âĸ
âĸ
âĸ
âĸ
acidic urine upto mark U
and reagent is added upto mark R.
Tube is shaken well by inversion.
Stopper the tube.
Keep in standing erect position for 18-24 hours for the
precipitate to settle down.
Reading of the length of ppt is taken indicated by
markings present over the tube.
Albumin is expressed in gm/L of urine.
46. ī When test done on 24 hours urine sample,
quantity of urine passed per day may be
calculated by
ī Dividing quantity of albumin per litre by total
quantity of urine passed in 24 hours in litre.
47. Microalbuminuria
ī Urinary albumin excretion between 30-300
mg/day.
ī Cannot be detected by dipstick methods.
ī Strong predictor of development of diabetic
nephropathy.
ī Can be detected 10-15 years before
development of diabetic nephropathy.
ī Significant risk marker of cardiovascular ds.
49. Bence Jones proteins
ī BJ protein is abnormal LMW globulin consisting of
light chains of Ig either Lambda or Kappa chains.
ī Characteristic feature- PPT at 400 C to 600 C and
redissolves at higher temperature (1000c) &
reappears when the urine is cooled.
ī Conditions a/w BJ proteinuria:
ī Multiple myeloma
ī Plasmacytoma
ī Waldesnstrom macroglobinaemia
50. Detection of Bence-Jones protein
ī Take 5ml urine in a test tube.
ī If the urine is cloudy, than filter it with filter paper.
ī If the reaction is alkaline of urine than do it acidic by adding
ī
ī
ī
ī
ī
a few drops of 25% acetic acid.
Than set the test tube in a water bath.
Heat in water bath for 15 minutes.
If the Bence -Jones Protein is present in urine then
precipitate forms between temperature of 40°C -60°C.
But when temperature is raised to 850 -100°C, precipitate
disappears.
When the temperature is decreased to 60°C, precipitate
reappears.
0
51. SUGARS IN URINE
ī This is a non-specific test useful for
semiquantitation of marked glucosuria.
ī Benedictâs qualitative test
ī Principle- Aldehyde group of reducing sugar
reduces Cupric ions in Benedictâs reagent to
cuprous oxide.
ī Detects all sugars except sucrose.
52. ī The final color of the solution depends on how
much of this precipitate was formed, and
therefore the color gives an indication of how
much reducing sugar was present.
ī Increasing amounts of reducing sugar
ī Green yellow orange red
53. Components of Benedictâs reagent
ī Sodium carbonate- 100 gm (Provides alkaline
conditions which are required for the redox
reaction)
ī Sodium citrate- 173 gm (complexes with the
copper (II) ions so that they do not deteriorate to
copper(I) ions during storage)
ī Copper sulphate- 17.3 gm
54. ī Procedure
ī Take 5ml of Benedictâs reagent
ī Boil for 3 â 5 minutes
ī Add 0.5ml (8 drops)of urine.
ī Boil for 2 minutes.
ī Cool and note the colour.
55. ī Recording results
ī The color varies from blue through green â yellow-
orange- brick red.
ī Negative
ī Trace
ī 1+
ī 2+
ī 3+
ī 4+
No change in color.
Greenish blue
Greenish yellow (0.5% sugar)
Yellow (1% sugar)
Orange precipitate (1.5% sugar)
Brick red precipitate (2% sugar)
58. COLORIMETRIC REAGENT STRIP
TEST
ī Principle: this test is based on a double
sequential enzyme reaction.
ī One enzyme, glucose oxidase, catalyzes the
formation of gluconic acid and hydrogen
peroxide from the oxidation of glucose.
ī A second enzyme, peroxides catalyzes the
reaction of hydrogen peroxide with potassium
iodide chromogen to oxidize the chromogen to
colors ranging from green to brown.
65. ī Rotheraâs Test for Acetone and
Acetoacetic Acid:
ī Principle:
ī Acetone and acetoacetic acid develops purple
coloured complex with sodium nitroprusside in
alkaline medium.
ī Hartâs test
ī For detection of beta-hydroxybutiric acid.
66. ī Rotheraâs Test for Acetone and
Acetoacetic Acid:
ī Procedure:
ī Take 5ml of urine in a test tube and saturate it
ī
ī
ī
ī
with ammonium sulphate.
Add 1 crystal of sodium nitroprusside.
Mix.
Run liquid ammonia carefully at the side of the
tube so as to form a layer on top of the saturated
urine.
POSITIVE- Formation of purple ring at junction
of two fluids.
67.
68. OCCULT BLOOD IN URINE:
ī Red blood cells / haemoglobin.
ī Haematuria- when 5 or more intact
RBCs/HPF.
69. Causes of Haemoglobinuria
ī Malaria- black water fever.
ī Hemolytic streptococcal septicaemia.
ī Incompatible blood transfusion.
ī Drugs- Sulphonamides, phenylhydralazine.
ī PNH
71. Benzidine Test
ī PRINCIPLE
ī The peroxidase activity of hemoglobin decomposes
hydrogen peroxide releasing nascent oxygen which
in turn oxidizes benzidine to give blue color.
ī REAGENTS
ī A: Saturated solution of benzidine in glacial acetic
acid
ī B: Hydrogen peroxide
72. Benzidine Test
ī PROCEDURE
ī Add 2 ml of urine in test tube.
ī Add 2ml of 1% Benzidine solution in acetic acid.
ī Shake well.
ī Add 2ml of hydrogen peroxide.
ī Mix and observe for a change in color.
ī Positive result: Green or blue color.
(Hematuria)
75. Bile salts
ī Primary bile acids
ī Cholic acid and chenodeoxycholic acid (CDCA)-
synthesized from cholesterol in the liver,
conjugated with glycine or taurine, and secreted
into the bile.
ī Secondary bile acids
ī Deoxycholate and lithocholate, are formed in the
colon as bacterial metabolites of the primary bile
acids.
ī Sodium taurocholate and sodium glycocholate are
76. Tests for detection of bile salts
ī Hay Test
ī Principle:
ī Bile salts when present decreases surface tension
of urine.
ī Procedure:
ī Take 10 ml of urine in beaker.
ī Sprinkle dry sulphur powder on the surface of the
urine
ī Results:
ī If bile salts are present they sink to the bottom.
77. Bile pigments
ī Normal urineī Urochrome
ī Traces of Urobilin
ī Abnormal urine
ī Bilirubin
ī Urobilinogen
ī Biliverdin
ī Urobilin
78. Fouchets Test/Harrisonâs spot test
ī FOUCHETS REAGENT
ī Trichloroacetic acid â 25 gms
ī Distilled water - 100 ml
ī 10% Ferric chloride solution â 10 ml.
ī Principle:
ī Barium chloride added to urine combines with sulphate
radicals in urine to form precipitate of barium phosphate. If
bile pigments are present in urine, they will adhere to these
large molecules. Ferric chloride present in fouchet reagent
then oxidizes yellow bilirubin in presence of trichloroacetic
acid to green bilverdin.
79. Fouchets Test/Harrisonâs spot
test:
ī PROCEDURE
ī Place 5 ml of acidified urine in a test tube.
ī Add 5ml of 10 % barium chloride.
ī Mix and filter through filter paper.
ī Let the paper dry.
ī Add 1-2 drops of Fouchetâs reagent to the ppt
on filter paper.
ī RESULT: A green color indicates the
presence of bilirubin.
80. Ehrlichâs test for urobilinogen
ī Principleī Urobilinogen reacts with p-dimethylamino-
benzaldehyde to form red colour.
ī Intensity of red colour is proportional to the
concentration of urobilinogen in urine.
ī Reagentsī P-dimethylaminobenzaldehyde
ī HCL
ī DW
81. Ehrlichâs test for urobilinogen
ī Procedure
ī Add 1ml of Ehrlichâs reagent to 10 ml of urine in
test tube.
ī Mix by inversion.
ī Let stand for 5 minutes.
ī RESULT
ī Pink- Normal
ī Dark red colour- Positive for urobilinogen.
83. LABELLING
ī Sample container-for identification of
sample.
ī Cytology requisition form-for
identification of individual patient
sample.
84. CENTRIFUGATION
Basically of 2 typesī I
Normal.
ī II
Cytospin-A device that spins cells in a fluid
suspension .
ī
Drawbacks-distortion of cellular morphology due to
air drying artifacts and loss of cells by absorption
of fluid into the filter card.
ī
In this process, urine sample is taken in a conical
tube and centrifuge at a rate of 2000rpm for 1015
minutes.
85. PAPANICOLAOU STAIN
ī Done by two methods-
1
Automated stainer-- large scale slides.
Takes 30 minutes for
staining.
2
Manual staining using copplin jarFor small scale slides.
Takes less than 7 minutes to
stain.
ī Staining objectivesI Well stained nuclear chromatin.
II Differential counterstaining i.e. staining the
86. Urine Cytology
ī INTERPRETATION-
1 Normal-Normal constituents of urine.
2 Abnormal-Any variation from normalI cellular components
II Acellular components.
CELLS derived fromī Urothelial and its variants.
ī Renal tubules.
ī Adjacent organs-like prostrate.
ī Cells extragenous to the urinary tract-RBCs.
88. C Crystals-Some are common in Acidic urine
-Some are common in Alkaline urine
D
Bacteria-Normal urine is free from bacteria.
E
Yeast.
F
Malignant cells.
G
Artifacts.
89. CELLS
Erythrocytes
ī usually appear as hourglass appearance.
ī presence of red cells 1-2RBCs/HPF is not
considered abnormal.
ī in hypotonic urine red cells swell up
causing
lysis -releasing Hb in urine-lysed cells are
referred as ghost cells.
ī when the red cells are swollen/crenated
sometimes mistaken for WBCs and yeast
cells
90.
91. Leucocytes
ī Normal up to 1-2 WBCs/HPF.
ī Larger than red cells and smaller than renal
epithelial cells.
ī Usually spherical- singly/clumps.
ī Mostly neutrophils-presence of characteristics
granules and lobulation.
ī Addition of 2%acetic acid to slide accentuated the
nuclei of cells.
ī Presence of many white cells in clumps is strongly
suggestive of acute urinary tract infection.
92.
93.
94. Epithelial cells
ī Any site in genitourinary tract from PCT to the urethra or
from the vagina.
ī Normally a few cells from these sites can be found.
ī A marked increase indicates inflammation of that
proportion of urinary tract from which the cell is derived.
TYPES
ī Renal tubular
ī Transitional
ī Squamous
95. Renal tubular epi. cells
ī Larger than white cells
ī Large round nucleus.
ī May flat/ cuboidal/ columnar.
ī Increase no indicates tubular
damage.
96. Transitional epithelial cells
ī 2 to 4 times larger
than white cells.
ī Round/ pear shaped/
may have tail like
projection.
ī Line the urinary tract
from pelvis of kidney
to upper portion of
urethra.
98. Crystals
ī Usually not found in fresh urine but appear
when urine strands for a while.
ī Many of crystal found in urine have little
clinical significance except in case of
metabolic disorders.
ī Crystals are identified by their appearance
and their solubility characteristics.
TYPESī Acidic urine crystals
ī Alkaline urine crystals
101. Uric acid crystals
ī Most
characteristics
form are diamond
or rhombic prism.
ī Presence of uric
acid crystals in
urine is a normal
appearance.
ī Increase in-gout
-AFI
-Chronic nephritis
-high purine
metabolism
102. Calcium oxalate crystals
ī Octahedral or envelope
shaped crystal
ī Can be present in normal
urine after ingestion of
various oxalate rich foods.
ī Pathological- DM
-Liver disease
-Severe chronic
renal
disease
103. Amorphous urates
ī Urates salts of sod, pot.
and calcium
ī Having a granular
appearance
ī Present in urine as non
crystalline amorphous
forms.
ī No clinical significance.
104. Hippuric acid crystals
ī Elongated prism like.
ī Rarely seen in urine.
ī No clinical significance.
105. Cystine crystals
ī Refractile
hexagonal plate
swith equal or
unequal sides.
ī Frequently have
layered or
laminated
appearance.
ī Soluble in
ammonia.
ī Can be detected
chemically by
Sod-cyanide-sod.
106. Leucine crystals
ī Highly refractile
having spheroid with
radical and concentric
striations.
ī Clinically very
significant.
ī Maple syrup disease
ī Serious liver disease
107. Tyrosine crystals
ī very fine needle
likes occurring in
sheaves or
clusters.
ī Clinically
significance
ī Severe liver
disease
ī tyrosinosis
109. Sulfa drugs crystals
ī precipitate as
sheets of
needles
usually with
eccentric
binding
ī May be history
of sulfa drugs
medication
110. Alkaline urine crystals
Triple phosphates crystalsī prism like with three to
six sides
ī Frequently found in
normal urine
ī Pathological
-chronic pyelitis
-cystitis
-enlarged
prostate
113. Casts
ī Presence of casts are frequently associated with
proteinuria.
ī Have nearly parallel sides with rounded or blunts
ends.
ī Always renal in origin and indicates intrinsic
renal disease
ī Casts are more or less circular with thicker in
middle.
116. Granular casts
ī degeneration of
cellular casts or
direct
aggregation of
serum proteins.
ī Almost always
indicate
significant renal
disease.
ī May be fine
granular or
coarse granular
casts.
117. Epithelial cells casts
ī result as statis and desquamation of renal tubular
epithelial cells.
ī Indicates tubular injury.
119. Fatty casts
ī Appear as a
few fat
droplets or
compose
almost
entirely of fat
droplets of
various
sizes.
ī Found in
fatty
degeneration
of tubular
epithelial.
120. Hyaline cast
ī Damage to glomerular capillary membrane, fever,
orthostatic proteinuria, and emotional stress or
strenuous exercise.
Flocculation- formation of fluffy masses/aggregates held together by weak physical attractionsPrecipitation- formation of solidppt in solution during chemical reaction
If alkaline urine add acetic acid
Glucose is oxidized to gluconic acid and hydrogen peroxide by glucose oxidase. Hydrogen peroxide reacts with o-dianisidine in the presence of peroxidase to form a colored product. Oxidized o-dianisidine reacts with sulfuric acid to form a more stable colored product. Theintensity of the pink color measured at 540 nm is proportional to the original glucose concentration