about Urine Examination in Pathology

1. Urine examination (practical)

2. PA 23.1 : Describe abnormal urinary findings
in disease states and describe common urinary
abnormalities in clinical specimen

3. SLO -
• To learn how to perform urinary examination
• To learn about common urinary findings in
various disease states.

4. Urine Examination
(Physical and Chemical)
Routine examination of urine is discussed under
four headings:
A. Adequacy of specimen
B. Physical/gross examination
C. Chemical examination
D. Microscopic examination

5. ▪ ADEQUACY OF SPECIMEN
The specimen is to be collected in a clean container,
and properly labelled with name of the patient, age
and sex, date, hospital no. and time of collection.
▪ Specimen Collection
For routine specimen, a clean glass tube or capped jar.
A mid-stream sample is preferable i.e. first part of
urine is discarded and mid-stream sample is collected.
For 24-hour sample, collection of urine is started in
the morning at 8 AM and subsequent samples are
collected till next day 8 AM but either the first or the
last sample should not be included.
For Urine Culture, sterile containers are needed.

6. Methods of Preservation of Urine -
• Urine should be examined fresh, But if it has
to be delayed then following preservation
procedures can be followed:
i. Refrigeration at 4°C.
ii. Toluene
iii. Formalin
iv. Thymol
v. Acids: Hydrochloric acid, sulphuric acid
and boric acid

7. PHYSICAL EXAMINATION
Volume
• Normal daily urinary volume: 700-2500 ml (average 1200 ml)
Abnormalities:
i) Nocturia: >500 ml excretion at night.
ii) Polyuria: >2500 ml excretion in 24 hrs.
Physiological - excess water intake, in winter season
Pathological - diabetes insipidus, diabetes mellitus
iii) Oliguria: <500 ml of urine is passed in 24 hours.
Causes- low water intake, dehydration, renal ischaemia.
iv) Anuria: almost complete suppression of urine (< 150 ml) in 24hrs
Causes- renal stones, tumours, or renal ischaemia.

8. Colour
• Normal- clear, pale or straw-coloured due to pigment
urochrome.
• Various colour changes in urine:
i) Colourless - diabetes mellitus, diabetes insipidus,
excess intake of water.
ii) Deep amber - good muscular exercise, high fever.
iii) Orange - increased urobilinogen, concentrated urine.
iv) Smoky – blood(scanty), vitaminB12, aniline dye.
v) Red - haematuria, haemoglobinuria.
vi) Brown due to bile.
vii) Milky due to pus, fat.
viii) Green due to putrefied sample, phenol poisoning.

9. Odour
• Normal - faint aromatic odour.
Abnormal odours:
i) Pungent due to ammonia produced by bacterial
contamination.
ii) Putrid due to UTI.
iii) Fruity due to ketoacidosis.
iv) Mousy due to phenylketonuria.

10. • Reaction/pH
• Ability of the kidney to maintain H+ ion concentration in
extracellular fluid and plasma.
• Measured by - pH indicator paper or by electronic pH meter.
• Normal pH - slightly acidic , range 4.6-7.0 (average 6.0).
• Abnormal Ph may be due to:
Acidic urine (pH<7.0):
i. High protein intake, e.g. meat.
ii. Ingestion of acidic fruits.
iii. Respiratory and metabolic acidosis.
iv. UTI by E. coli.
Alkaline urine (pH>7.0):
i. Citrus fruits, certain vegetables.
ii. Respiratory and metabolic alkalosis.
iii. UTI by Proteus, Pseudomonas.

11. Specific Gravity
• Specific gravity is used to measure the concentrating and
diluting power of the kidneys.
• It depends upon the concentration of various
particles/solutes in the urine.
• It can be measured by urinometer, refractometer or reagent
strips

12. Urinometer Procedure
i. Fill urinometer container 3/4th with urine.
ii. Insert urinometer into it so that it floats in
urine without touching the wall and bottom of
container
iii. Read the graduation on the arm of
urinometer at lower urinary meniscus.
iv. Add or subtract 0.001 from the final
reading for each 3°C above or below the
calibration temperature respectively marked on
the urinometer.

13. Significance of Specific Gravity
• Normal - 1.003 to 1.030.
• Low specific gravity urine occurs in:
i. Excess water intake
ii. Diabetes insipidus
• High specific gravity urine is seen in:
i. Dehydration
ii. Albuminuria
iii. Glycosuria.
• Fixed specific gravity (1.010) of urine is seen in:
i. ADH deficiency
ii. Chronic kidney disease (CKD).

14. CHEMICAL EXAMINATION
Chemical constituents frequently tested in urine
are:
• proteins
• glucose
• ketones
• bile derivatives
• blood

15. Proteinuria
• If urine is not clear, it should be filtered or
centrifuged before testing for proteins.
• Urine may be tested for proteinuria by
qualitative tests and quantitative methods.
• Qualitative Tests for Proteinuria
1. Heat and acetic acid test
2. Sulfosalicylic acid test
3. Heller’s test
4. Reagent strip method.

16. Heat and Acetic Acid Test
Principle- Heat causes coagulation of proteins.
Procedure-
❖ Take a 10 ml test tube.
❖ Fill 2/3rd with urine.
❖ Acidify urine by adding a few drops of 3% glacial acetic acid.
❖ Boil upper portion for 2 minutes (lower part acts as control).
❖ If precipitation or turbidity appears, add a few drops of 10% acetic acid.
Interpretation:
• If turbidity disappears on addition of acetic acid, it is due to phosphates; if it
persists then it is due to proteins.
No cloudiness = negative
Faint cloudiness = traces (< 0.1 g/dl)
Cloudiness without granularity = +1(0.1 g/dl)
Granular cloudiness = +2(0.1-0.2 g/dl)
Precipitation and flocculation = +3(0.2-0.4 g/dl)
Thick solid precipitation = +4(0.5 g/dl)

17. Heat and acetic acid test for proteinuria. Note the method of
holding the tube from the bottom while heating the upper part.

18. Reagent Strip Method
• Bromophenol coated strip is dipped in urine.
• Change in colour of strip indicates presence of proteins in
urine; colour change is compared with the colour chart
provided on the bottle containing strips and it gives
semiquantitative grading of proteinuria

19. Quantitative Estimation of Proteins in Urine
Methods:
1. Esbach’s albuminometer method
2. Turbidimetric method.

20. Esbach’s Albuminometer Method:
• Fill the albuminometer with urine up to mark U.
• Add Esbach’s reagent up to mark R .
Esbach’s Composition : 10 gm picric acid & 20 gm citric
acid in 1000 ml distilled water
• Stopper the tube, mix it and let it stand for 24 hours.
• Take the reading from the level of precipitation in the
albuminometer tube and divide it by 10 to get the
percentage of proteins.

21. Esbach’s albuminometer for quantitative estimation of proteins

22. Bence Jones Proteinuria
• Bence Jones (BJ) proteins are light chains of
γ-globulin. These are excreted in multiple
myeloma and other paraproteinaemias.
• BJ proteins are precipitated at lower
temperature (56°C), disappear on further
heating above 90°C but reappear on cooling
to lower temperature again.

23. Glucosuria
• Glucose is by far the most important of the sugars
which may appear in urine.
• Tests for glucosuria may be qualitative or
quantitative.
Qualitative Tests
These are as under:
1. Benedict’s test
2. Reagent strip test

24. Benedict’s Test :
Cupric (Cu2+) ion is reduced by glucose to
cuprous(Cu1+) oxide and a coloured precipitate is
formed.
Procedure:
❖ Take 5 ml of Benedict’s qualitative reagent in a 20
ml test tube.
❖ Add 8 drops (or 0.5 ml) of urine.
❖ Heat to boiling for 2 minutes .
❖ Cool in water bath or in running tap water and
look for colour change and precipitation.

25. Interpretation
• No change of blue colour = Negative
• Greenish colour = traces (< 0.5 g/dl)
• Green/cloudy green ppt = +1 (0.5-1 g/dl)
• Yellow ppt = +2 (1-1.5 g/dl)
• Orange ppt = +3 (1.5-2 g/dl)
• Brick red ppt = +4 (> 2 g/dl)

26. Since Benedict’s test is for reducing
substances excreted in the urine, the test is
positive for all reducing sugars (glucose,
fructose, maltose, lactose)
Other reducing substances (e.g. ascorbic acid,
salicylates, PAS, antitubercular drugs such as
PAS, isoniazid) also give False positive test.

27. Reagent Strip Test
▪ These strips are coated with glucose oxidase
and the test is based on enzymatic reaction.
▪ This test is specific for glucose.
▪ The strip is dipped in urine for 10 seconds. If
there is change in colour of strip, it indicates
presence of glucose.
▪ The colour change is matched with standard
colour chart provided on the label of the
reagent strip bottle

29. Ketonuria
▪ Ketones are products of incomplete fat
metabolism.
▪ The three ketone bodies excreted in urine are:
acetoacetic acid (20%), acetone (2%), and
β-hydroxybutyric acid (78%).
Tests for Ketonuria
1. Rothera’s test
2. Gerhardt’s test
3. Reagent strip test

30. Rothera’s Test
• Principle : Ketone bodies (acetone and acetoacetic
acid) combine with alkaline solution of sodium
nitroprusside forming purple complex.
Procedure :
❖ Take 5 ml of urine in a test tube.
❖ Saturate it with solid ammonium sulphate salt; it will
sediment to the bottom of the tube when saturated.
❖ Add a few crystals of sodium nitroprusside and shake.
❖ Add liquor ammonia from the side of test tube.
❖ Interpretation : Appearance of purple coloured ring at
the junction indicates presence of ketone bodies

31. Rothera’s test for ketone bodies in urine showing purple
coloured ring in positive test.

32. Tests for Bile Salts
▪ Bile salts excreted in urine are cholic acid and
chenodeoxycholic acid.
▪ Tests - Hay’s test and Reagent strip method.
Hay’s Test
Principle: If bile salts are present in urine, they
lower the surface tension of the urine.
Procedure :
❖ Fill a 50 or 100 ml beaker 2/3rd to 3/4th with
urine.
❖ Sprinkle finely powdered sulphur powder over it.
Interpretation : If bile salts are present in the urine,
then sulphur powder sinks, otherwise it floats.

33. Hay’s test for bile salts in urine. The test is positive in beaker in the
centre contrasted with negative control in beaker on right side.

34. Blood in Urine
Tests for detection of blood in urine are as under:
▪ Benzidine test
▪ Orthotoluidine test
▪ Reagent strip test.

35. • Benzidine Test
Procedure : 2ml urine + 2ml saturated sol. of
benzidine with glacial acetic acid -> add 1 ml H2
O2
->
blue colour
Benzidine is carcinogenic and not commonly used.
• Orthotoluidine Test
Procedure: 2ml urine + 1 ml orthotoluidine in glacial
acetic acid -> Add few drops of H2
O2
-> Blue or green
colour
• Reagent Strip Test :
The reagent strip is coated with orthotoluidine.
Dip the strip in urine -> blue colour

36. Microscopy
PREPARATION OF SEDIMENT :
• Take 5-10 ml of urine in a centrifuge tube.
• Centrifuge for 5 minutes at 3000 rpm.
• Discard the supernatant.
• Resuspend the deposit in 0.5-1 ml of urine left.
• Place a drop of this on a clean glass slide.
• Place a coverslip over it and examine it under the
microscope.

37. • Microscopy is routinely done under reduced
light using the light microscope.
• This is done by keeping the condenser low
with partial closure of diaphragm.
• First examine it under low power objective,
then under high power and keep on changing
the fine adjustment in order to visualise the
sediments in different planes and then report
as number of cells/HPF (high power field).

38. Following categories of constituents are
frequently reported in the urine on microscopic
examination:
1. Cells (RBCs, WBCs, epithelial cells)
2. Casts
3. Crystals
4. Miscellaneous structures

39. Cells in Urine
RBCs : These appear as pale or yellowish,
biconcave, double contoured, disc-like
structures
Normal: 0-2 RBCs/HPF.
Microscopic hematuria is presence of >3
RBCs/HPF in a visibly normal coloured urine.
Gross hematuria refers to visibly hemorrhagic or
red coloured urine

40. Excess RBCs are seen in urine in the following conditions:
Physiological
i. Following severe exercise
ii. Smoking
iii. Lumbar lordosis
iv. In menstruating females
Pathological
i. Renal stones
ii. Renal tumours
iii. Nephritic syndrome
iv. Polycystic kidney
v. UTI
vi. Trauma

41. WBCs (Leucocytes/Pus cells)
❖ These appear as round granular 12-14 μm in
diameter.
❖ In fresh urine nuclear details are well
visualised.
• Significance : Normally 0-4 WBCs/HPF may be
present.

42. Excess WBCs (Pus cells) are seen in urine in
following conditions:
i. UTI
ii. Cystitis
iii. Prostatitis
iv. Chronic pyelonephritis
v. Renal stones
vi. Renal tumours

43. WBC (pus cells) in urine
(WBC/Pus cells)

44. Epithelial Cells:
• These are round to polygonal cells with a
round to oval, small to large nucleus.
• Can be squamous epithelial cells, tubular
cells and transitional cells
• Normally a few epithelial cells are seen in
normal urine, more common in females and
reflect normal sloughing of these cells .
• When these cells are present in large number
along with WBCs, they are indicative of
inflammation.

45. Epithelial cells in urine
Epithelial cells

46. Casts in Urine
• Urinary casts have parallel margins and take the contour of the
portion of tubule in which they are formed
• These are formed due to moulding of solidified proteins in renal
tubules
• Mostly cylindrical in shape with rounded ends.
• The basic composition of casts is Tamm Horsfall protein which is
secreted by tubular cells.
• Depending upon the content, casts are of following types :
i. Hyaline cast
ii. Red cell cast
iii. Leucocyte cast
iv. Granular cast
v. Waxy cast
vi. Fatty cast
vii. Epithelial cast
viii. Pigment cast

48. Crystals in Urine
Crystals in Acidic Urine :
i. Calcium oxalate
ii. Uric acid
iii. Amorphous urate
iv. Tyrosine
v. Cystine
vi. Cholesterol

50. Crystals in Alkaline Urine
i. Amorphous phosphates
ii. Triple phosphates
iii. Calcium carbonates
iv. Ammonium biurates

52. Thank You