2. URINE EXAMINATION is an important lab test as many
different diseases can display abnormalities in the urine .
Basic urinalysis consists of gross examination of the
urine, as well as a dipstick analysis for blood, white blood
cells, sugar, and other substances.
A microscopic analysis of urine may be necessary in
many cases. This is done to detect cellular elements, casts,
and crystals.
6. 2)PROPER SPECIMEN –
Methods of urine collection
Midstream Clean Catch Specimen
Catheter Specimen
Plastic Bag Specimen- infants
Supra pubic aspiration
Three glass Specimen-prostate infection
7. Collection of urine for
A) Routine examination
containers should be:
wide mouthed , Clean and leak proof.
Break-resistant , Disposable , clear material
Capacity of 50ml(routine) and 3lt. (24hr sample)
Amber coloured containers for light sensitiveAnalytes
15 ml of midstream sample
B) Culture examination
Midstream clean catch sample
Plated within 2hrs of collection (if refrigerated – 24hrs)
No preservatives
If single specimen is received for multiple tests then
bacteriological examination should be done first.
8. C) Proper Preservative
REFRIGERATION (mc)
Ideally the specimen should be examined within 2 hoursof voiding.
If delay is expected, can be refrigerated for maximum of 24hrs
4-6°C refrigeration upto 8hrs is best general preservation method
9. Preservatives for testing of
Steroids HCl + copper sulphate
VMA HCl
Porphyrin Sodium carbonate
CHEMICAL PRESERVATIVES –
Formalin 3 drop/100 ml ( formed elements)
Toluene 2 ml/100 ml (chemical substances)
Boric acid 0.5gm/60 ml (general preservative)
Thymol 1crystal/100m (sediments )
Conc. HCl (Adrenaline, nor-A, VMA, steroids)
10. D) Visible Signs Of Contamination
Effect of Storage on urine:
1.Decrease in clarity , foul smeling odour
Increase in pH.
2. Formation of crystals
3. Loss of ketone bodies
4. Decrease in glucose
5. Oxidation of bilirubin to biliverdin
6. Oxidation of urobilinogen to urobilin
7. Increase in nitrites
8. Disintegration of cellular elements
11.
12. • Color
• Clarity
• Odor
• Urine Volume
• Specific gravity
• Osmolality
Physical
parameters
14. COLOR PATHOLOGICAL NON PATHOLOGICAL
Pink to Red to Red -
Brown
Red blood cells
Haemoglobin
Myoglobin
Porphyrins
Beets(anthocynin)
Methyldopa
YELLOW TO ORANGE Bilirubin
Urobilin
Concentrated urine
Rifampicin
Acriflavine
Carrots
15. COLOR PATHOLOGICAL NON PATHOLOGICAL
Brown to Black Methaemoglobin
Rhabdomyolysis
Homogentisic acid
Melanin
Iron compounds
Levodopa
Chloroquine
Metronidazole
Blue to Green Biliverdin
Pseudomonas
Infection
Food dyes
Additives
Amitryptiline
Cimetidine
Methylene blue
Vit B complex
18. Freshly voided urine- Typical aromatic odour (Volatile organic acids)
On standing- Conversion of urea ammonia– Faint ammoniacal odour
19. Volume of only the 24 hour specimen of urine is to be measured .
Average 24hr urinary output in adults is 600 to 2000ml
Called as Amount Causes
1. Polyuria
>2000ml in 24
hours
•Diabetes mellitus,
•Diabetes insipidus
•Chronic renal failure
•Diuretic therapy
2.
Oliguria
< 500 ml in 24
hours
•Fever
•Dehydration
•Congestive heart failure
•Acute glomerulonephritis
3. Anuria
< 100 ml or
NO output in
24 hours
•Complete urinary tract obstruction
•Acute tubular necrosis
•Acute glomerulonephritis
20. Ratio of weight of a volume of urine to the weight of
the same volume of distilled water at a constant
temperature.
Main contributors - UREA (20%) and NaCl (25%)
Measures the concentrating and diluting power of
kidney.
Concentrating ability of kidney is one of the first
function to be lost as a result of tubular damage.
21. Specific gravity of distilled water= 1.000
Normal sp.gr. of urine
Random sample 1.003 to 1.030 ( at least >
1.023)
24 hr sample with normal fluid
intake
1.016 to 1.022
After a standard water load < 1.007
Abnormal sp.gr. of urine
HYPO- STHENURIA Consistently <
1.007
DI, GN, pyelonephritis
ISO- STHENURIA Fixed at 1.010 Severe Renal Damage
HYPER-STHENURIA Higher than
normal
Fever, dehydration,
CHF, adrenal
insufficiency, DM,
nephrotic syn.
22. Methods of Specific Gravity
measurement
INDIRECT METHODS
1. Reagent strip method
2. Urinometer
DIRECT METHODS
3. Refractometer
4. Falling drop method
M/C EMPLOYED
23. These measure the ion concentration in urine
Contents of a reagent strip pad-
(1) polyelectrolyte (2) indicator substance (3) buffer
PRINCIPLE: pKa change of pretreated polyelectrolyte
in relation to the ionic concentration of urine.
Higher the concentration of urine, lower the pKa as
pKa refers to the proton donating capacity of an
acid and stronger the acid, lower the pKa.
ions of
urine
Contact
with
reagent
pad
Acid groups
dissociate
Release H+
Change of
pH
24. The strips have colour pads with increment of reading of
0.005
Advantages:
1. This method is not affected by the high
amounts of protein and sugar and so there is
no need for the correction of value.
2. This is a method unaffected by
Temperature changes.
Disadvantages:
• False readings may be obtained if there is a
run-over from adjacent reagent area in
excessively wetted strips.
25. Indicates the number of particles of solute per unit of solution
Normal fluid intake- 500 to 850 mOsm/kg water.
800 to 1400 mOsm/kg water in dehydration,
After a period of dehydration, the osmolality of the urine should be
three to four times that of the plasma
o 40 to 80 mOsm/kg water during water diuresis.
o 285 mosm/kg in terminal renal failure
Measured by freezing point depression using osmometer.
A solution containing 1osmol or 1000 mOsm/kg water depresses
the freezing point 1.86° C below the freezing point of water
26.
27. REAGENT STRIPS
Primary method used for the
chemical examination of
urine.
Reagent strips are dip- and -
read strips for in vitro
diagnostic use only for
testing various contents in
urine.
It is measured by
comparison of test paper
attached to plastic strip
with color of chart blocks
printed on vial label.
28. Testing
Test urine as soon as possible after receipt.
Test a well-mixed, unspun urine sample.
Urine samples must be at room temperature before testing.
Do not use reagent strips in the presence of volatile acids or alkaline fumes
Dip reagent strip into urine briefly—no longer than 1 second.
Follow exact timing recommendations for each chemical test.
Hold reagent strip close to the color chart, and read under good lighting.
Know sources of error, sensitivity, and specificity of each test on the
reagent strip.
Think! Make correlations between patient history and individual test, and
then follow through.
29.
30.
31. Advantages of Multistix Strip
Quick screening of urine chemistry
Reliable, specific and sensitive
Avoids use of various corrosive reagents, different type
of glass wares and other laboratory material required for
wet chemical testing of urine
It can be performed in uncentrifuged urine and doesn’t
require acidification
Less labour intensive and can be automated for large
laboratories.
Less chance of human error
32. Normal pH is 4.6 to 8.0 (avg -6)
Methods:
1. Reagent strip method
2. Litmus paper test
3. pH meter
Acidic urine Alkaline urine
Diabetes mellitus UTI by urea splitting organisms
(Proteus and Pseudomonas)
Starvation Severe vomiting
Fever Vegetarian diet (Citrus fruits)
UTI by E. coli Old ammoniacal urine sample
High protein diet Chronic renal failure
33. • Indicators methyl red and bromothymol blue give a range of
orange, green, and blue colours as the pH rises, permitting
estimation of pH values to within half a unit within the range of 5
to 9.
Problems:
If the strips get excessively wet, acid buffer from the protein
patch runs into the pH patch, causing it to become orange.
Results are not precise unless the test is performed on
freshly voided urine as with time, the pH increases
35. Normal- Up to 150 mg/24 hours , average protein
concentration varying from 2 to 10 mg/dl
Proteins from plasma (albumin) and Proteins from UT (
Tamm horsfall protein , IgA, Tubular epithelial cells ,
WBC’s, desquamated cells .
Very low maximal tubular rate of reabsorption
meaning increased filtration of protein quickly saturates
the reabsorptive mechanism.
Hence, detection of abnormal amounts of protein- IMP
indicator of renal disease
37. (> 4 gm/day)
Nephrotic syndrome (loss of albumin , globulin)
Acute and rapidy progressive glomerulonephritis
Chronic glomerulonephritis
Diabetic nephropathy, severe
Renal amyloidosis
Lupus nephritis
Toxemia of pregnancy
End Stage Renal Disease
Miscellaneous – malaria,malignant
hypertension,heavy metals,drugs
38. Qualitative tests
Reagent strip
Sulphosalicylic acid test
Heat Precipitation test
Heller’s nitric acid test
Quantitative tests
Esbach’s Albuminometer
Immunological method
There are other Quantitave methods available
but these have been found unsatisfactory.
39. Principle (protein error of indicators)-Reagent area of
strip is coated with an indicator (tetrabromophenol blue) and
buffered to an acid pH 3 which changes color in the presence
of proteins
indicator undergoes color change from yellow to shades of
green
Sensitive to albumin 5-10 mg/dl
Negative with Bence-Jones protein, Hb, Mb
40. Advantage:
• avoids false-positive reactions with organic iodides (e.g.
Radiograph contrast and tolbutamides or other drugs).
Drawbacks:
• Lack of sensitivity of the reagent strip to globulins
• False positive results can occur with highly pigmented urine,
quaternary ammonium compounds, fabric
softeners, chlorhexidine, and excessive leaching of the acid buffer
of the test strip by excessive wetting.
• Highly buffered alkaline urine will also provide false-positive
results.
41. Sulfosalicylic Acid Method
(Qualitative):
Principle- Proteins are denatured by organic acid and precipitate
out of sol.
Detects albumin, Hb, myoglobin,Bence Jones protein
Procedure:- Add 2-3 drops of 3% Sulphosalicylic acid in 2ml
urine, wait for 10 minutes and observe for degree of precipitation
43. GLUCOSE
Glycosuria occurs when the blood level is >180 - 200 mg/dl
Causes of Glycosuria:
1. Renal Glycosuria (due to low renal threshold)
e.g. Fanconi’s Syndrome
2. Gestational Glycosuria (Reduced Renal threshold)
3. Alimentary glycosuria
4. Endocrine causes-DM, Acromegaly, Cushing syndrome,
hyperthyroidism, pancreatic disease
Normally, 50 mg of disaccharides are excreted in 24 hours.
With severe Intestinal diseases like sprue or acute enteritis, this
level may rise to 250 mg or more.
44. Methods of Glucose estimation
1. REAGENT STRIP METHOD
2. COPPER REDUCTION TEST
• Benedict’s Test
• Clinitest Tablet
45. Reagent Strip method
• Principle: Based on a specific glucose oxidase and peroxidase
method, a double sequential enzyme reaction; reagent strips differ only
in the chromogen used. Glucose is oxidized by glucose oxidase with the
resultant formation of hydrogen peroxide and gluconic acid. Oxidation of
chromogen occurs in the presence of hydrogen peroxide and the enzyme
peroxidase with resultant color change
False negatives – ketones, ascorbic acid, salicylates, E.coli infection
False positive – Presence of oxidizing agents
46. Copper Reduction Tests: Done to cross verify
when
• false-negative is suspected due to ascorbic acid
• excretion of sugars other than glucose is suspected (infants)
Benedict’s Test
Principle-In benedict reagent cupric ion present in form of
copper sulphate (blue color) which is reduced by reducing
substance like glucose to cuprous oxide (brown red color)
47. Defect in carbohydrate metabolism or absorption, the body
compensates by metabolizing increasing amounts of fatty acids.
• 3 ketone bodies (products of incomplete fat metabolism ) present in
the urine are acetoacetic acid (20%), acetone (2%), and
3-hydroxybutyrate (about 78%).
Causes of Ketonuria
1. Diabetic Ketoacidosis: Type 1DM
2. Nondiabetic ketonuria: Acute febrile diseases, starvation,
Hyperemesis of pregnancy
3. Lactic Acidosis:Can coexist with Diabetes mellitus, Renal failure,
Liver disease.
48. Methods:
1. Reagent Strip method
2. Test tube nitroprusside method of Rothera
3. Nitroprusside Tablet test
4. Gerhardt ferric chloride test (Only for acetoacetic acid)
Commonly used method is the Reagent strip method,
detects as low as 10 mg of acetoacetic acid.
None of the tests mentioned above detects Beta-
Hydroxybutyric acid
49. Principle: Based on nitroprusside reaction for ketone.
• The chemo strip reagent strip contain sodium nitroprusside
buffer and glycine which reacts with acetoacetic acid in
presence of alkaline medium to form violet dye.
• Sensitivity: 10 mg/dl of acetoacetic acid and 70 mg/dl of
acetone.
50. Rothera’s Test
Principle -Acetone & acetoacetic acid
react with sodium nitroprusside in
the presence of alkali to produce
purple colour ring.
Method- take 5ml of urine in a test
tube & saturate it with ammonium
sulphate. Then add one crystal of
sodium nitroprusside. Then gently
add liquor ammonia along the sides
of the test tube.
Sensitivity - 1-5 mg/dl AAA, 10-15
mg/dl Acetone
51. • Gross hematuria: visible to the naked eye
• Microscopic hematuria: 3 or >3RBCs/HPF
• Reagent strip method is more sensitive than urine microscopy .Yet, it is
better to confirm this by microscopy.
Causes of Hematuria
1. Diseases of Urinary tract:
Glomerular diseases: Glomerulonephritis, Berger’s disease, lupus, HSP
Non-glomerular disease: Calculus, tumour, TB,pyelonephritis etc
2. Hematological conditions: Coagulation disorders, SCD
Presence of Red cell casts and proteinuria along with
hematuria suggests glomerular cause of hematuria
52. Principle:
Liberation of oxygen from peroxide in the reagent strip by
peroxidase like activity of hemoglobin, lysed erythrocytes or
myoglobin
Detect 0.3-0.5 mg/dl
• Haemoglobin catalyses the oxidation of chromogen to
produce green colour which is read at 60 seconds.
• If green spot is present then intact RBCs are present
• If uniform green colour is present then it indicates the
presence of haemoglobin or myoglobin.
53. • Breakdown product of Hemoglobin.
• Normal adult urine has only 0.02 mg/dl of Bilirubin
• Increased urinary Bilirubin occurs in-
1. Obstruction to bile outflow from liver
2. Hepatocellular disease with decreased excretion of
conjugated Bilirubin in bile
* Presence of Bilirubin in urine means presence of only
CONJUGATED BILIRUBIN
54. Methods:
1. Reagent Strip method
2. Diazo tablet method
3. Wash-through tablet method
• when urine is kept for a longer period of time(especially when
exposed to light), bilirubin glucuronide gets converted to free
bilirubin which isn’t picked up accurately on the Reagent strip
but is seen with the Tablet test
• Hence, Tablet tests are confirmatory tests .
The diazo test reacts positively to bilirubin in amounts as low as
0.05 to 0.1 mg per decilitre.
55. Principle: The test is based on coupling reaction of bilirubin with
diazonium salt in strongly acidic medium.
• Chemo strip reagent: Contains 6-dichlorobenzene diazonium
tetraflouroborate as salt which changes color from pink to violet at
30 -60 seconds. Detects 0.5 mg/dl of bilirubin
False negative-Exposure to light, ascorbic acid
False positives – Coloured metabolites of drugs(Rifampicin and
chlorpromazine
56. Bile Salts (Hay’s Sulphur powder test):
• Bile salts have the property of decreasing surface tension.
• Hence, sulphur powder sprinkled to urine will settle down
if bile salts are present in urine.
Bile Pigments (Fouchet’s Test):
• Bile pigments adhere to the precipitates of barium
sulphate.
• On addition of Fouchet’s reagent, ferric chloride in the
presence of trichloroacetic acid oxidizes yellow bilirubin
to green biliverdin
57. • Conjugated bilirubin in colon is acted upon by bacteria to form:
Urobilinogen which is excreted in the urine after the enterohepatic
circulation is complete.
• Normal output of urobilinogen in urine is 0.5 to 2.5 mg/24 hours
Causes Of Increased Urobilinogen
1. Hepatocellular damage.
2. Congestive heart failure (liver congestion)
3. Infection such as Cholangitis
4. Hemolysis (Urobilinogen without bilirubin
58. Methods:
1. REAGENT STRIP METHOD
2. EHLRICH’S ALDEHYDE METHOD
• For quantitative comparative purposes in the same patient,
a 2-hour test is used in which urine is collected from 2pm
to 4pm after lunch.
• This period coincides with heightened excretion of
urobilinogen, as the pH of the urine is more nearly
neutral.
59. Principle:
• Multistix :The test is based on modified Ehrlich’s
Aldehyde reaction .The test area is impregnated with p-
dimethyl aminobenzaldehyde with color ethanol which
reacts with urobilinogen in strongly acidic media to form
yellow shades of red brown.
not specific for urobilinogen .
Other porphobilinogen, indole, sterol can also give same
color development.
60. Chemo strip reagent: Test area is impregnated
with 4- methoxy benezene tetra flourate , which
couples with urobilinogenin an acid medium to form a red
azo dye .
can detect approximately 0.4 mg/dL.
specific for urobilinogen.
61. • Detects 70% positive results when compared to cultures
• E-coli only: 93% agreement with cultures
Most common organisms causing positive nitrite test:
1. Escherichia coli
2. Klebsiella
3. Enterobacter
4. Proteus
5. Staphylococcus
6. Pseudomonas
62. Principle:
• This is based on conversion of nitrate in urine to nitrite
by the action of bacteria
Nitrite+p-arsinilicacid Diazoniumcomplex
• This diazonium compound then couples with 1,2,3,4,
tetra hydrobenzoquinolin-3- ol to produce pink color.
Acidic pH
63. • Esterase activity- Marker for human neutrophils.
• Positive leukocyte esterase- Significant number of
Neutrophils either intact or lysed.
Reagent Strip Method:
The intracellular esterases (bacterial) catalyze hydrolysis of esters,
releasing a product that reacts with a diazonium salt to produce a
colored product.
