Introduction Urine analysis is a cornerstone diagnostic tool in clinical medicine. It is non-invasive, cost-effective, and provides significant insight into renal and systemic conditions. Among the three major aspects of urine examination — physical, chemical, and microscopic — the chemical examination is vital in detecting metabolic disturbances, renal disease, and infections. The most commonly used method in chemical analysis is the reagent strip test (urine dipstick), though manual and confirmatory methods like heat-acetic acid tests, SSA test, Benedict’s test, and Rothera’s test remain indispensable in certain contexts. Parameters of Chemical Examination The following components are routinely assessed: pH Proteins Glucose Ketones Bilirubin Bile salts Urobilinogen Blood Hemoglobin Myoglobin

1. URINE EXAMINATION
PHYSICAL AND
CHEMICAL

2. WHY STUDY URINE?
Urine is actually a “fluid biopsy” of the kidneys and
can provide a fountain of information about the
health of an individual. Urine is an ultrafiltrate of the
plasma, it can be used to evaluate and monitor
body homeostasis and many metabolic disease
processes.

3. ROUTINE URINE ANALYSIS IS MAINLY PERFORMED :
•To aid in the diagnosis of disease
•To screen for asymptomatic, congenital, or
hereditary disease
•To monitor disease progression
•To monitor therapy effectiveness or complications.

5. ROUTINE URINE ANALYSIS
The most commonly performed urine test is a routine
urinalysis test. A routine urinalysis evaluates three aspects
of the urine
1) its physical characteristics,
2) its chemical composition
3) the microscopic sediment elements (e.g., epithelial cells,
blood cells, casts, mucus) suspended in it.

6. URINE SPECIMEN TYPES

7. URINE COLLECTION TECHNIQUES

8. CONTAINER USED FOR URINE COLLECTION

9. PRESERVATION:
•All specimen for routine urinalysis should be examined within
one hour of collection to avoid degradation of chemical and
cellular material and prevent multiplication of bacteria. It should
be stored at 2 – 8◦ C in refrigerator.

10. PRESERVATIVE CONCENTRATION USAGE
TOLUNE 2ml/100 ml urine For measurements of chemicals
FORMALIN 3 drop/100ml urine Cytology
Thymol One small crystal per 100 ml of
urine
Sediment Preservation
Chloroform 5ml per 100 ml urine Form upper layer. It causes
changes in characteristics of
cellular sediment
Commercial preservative tablets.
These release formaldehyde
1 tablet/30 ml urine Concentration of formaldehyde
is controlled, so that it may not
interfere

11. • EXPECTED CHANGES IN THE COMPOSITION OF
URINE STORED AT ROOM TEMPERATURE
• Lysis of Red blood cell by hypotonic urine.
• Decomposition of casts.
• Bacterial multiplication.
• Decrease in glucose level, due to bacterial growth.
• Formation of ammonia from urea by action of bacteria
changes urine alkaline.

12. REASONS FOR URINE SPECIMEN REJECTION
• Unlabeled urine specimen container
• Mislabeled urine specimen
• Inappropriate urine collection technique or specimen type for test
requested (e.g., urine in diaper)
• Specimen not properly preserved during a time delay or
inappropriate urine preservative used
• Visibly contaminated urine (e.g., fecal material, toilet tissue)
• Insufficient volume of urine for test(s) requested

13. PHYSICAL PARAMETERS OF NORMAL URINE
• VOLUME : 600 - 2000 ml/24 hrs
• APPEARANCE : Clear
• COLOUR : Pale yellow or amber
• ODOR : Aromatic
• SPECIFIC GRAVITY : 1.003 - 1.030
• REACTION : ACIDIC (Ph : 4.5 - 8.0)

14. PHYSICAL EXAMINATION OF URINE:
•Volume:
1.Polyuria: > 2500 ml eg; Diabetes mellitus and Diabetes
insipidus etc
2.Oligouria : <500 ml eg: Shock and Acute nephritis etc
3.Anuria : Complete suppression of urine formation in
spite of high fluid intake eg. Renal failure.

15. Colour Condition Colour Condition
Colourless dilute urine eg
DI ,DM
Yellow green Biliverdin
Red Hematuria
Hemoglobinuria
Deep yellow with
yellow foam
Bilirubin
Dark brown or
black
Alkaptonuria(homo
gentisic acid)
Orange or
orange-brown
Urobilinigen,porph
obilinogen
Brown Myoglobinurea Milky-white Chyluria
Yellow Concentrated
urine,b complex
vitamins,nitrofurant
oin
Red or orange
fluorescence with
uv light
Porphyria
(Uroporphyrin,cop
roporphrin)
•COLOUR

16. Urine foam
due to high
bilirubin
Large amount
of urine foam
due to high
protein
concentration
Red Colour
Urine
Milky white
urine

17. •APPEARANCE
Cloudy - (Amorphous phosphates are present in alkaline urine
and amorphous urates are present in acidic urine)
•Turbid – mucus, leukocytes ,epithelial cell etc
• Milky - Fat & Chyle
• Smoky - Red Blood Cells

18. •Odor
• Sweet and fruity- Ketone bodies.
•Pungent smell- Bacteria due to formation of ammonia.
•Musty – Phenylketonuria.
•Burnt sugar- Maple syrup urine disease.

19. •REACTION AND PH
Acidic- High Protein, Acidic fruits, Respiratory acidosis,
Metabolic Acidosis, Diabeticketoacidosis, Severe Diarrhea, E.coli
infection etc.
Alkaline- High in Vegetables , Post prandial, respiratory and
metabolic alkalosis, Proteus and Pseudomonas infection etc.

20. .

21. SPECIFIC GRAVITY– Density of urine to density of distilled water at
similar temperature.
Determine concentrating and diluting power of Kidney
• HYPERSTHENURIA : (>1.010)Dehydration, Eclampsia, Proteinuria, lipoid nephrosis
• ISOSTHENENURIA :Excretion of urine at fixed specific gravity of 1.010 (CRF)
• HYPOASTHENURIA: (<1.010) Pyelonephritis, Hypertension, Diabetic insipidus, Protein
malnutrition, Diuretics Medicine, Alcohol ,Coffee.

22. •SPECIFIC GRAVITY DETERMINATION
1. URINOMETER METHOD:
Based on Buoyancy principle
• Solute concentration high, upthrust increases and
urinometer is pushed up.
•Solute concentration low, urinometer sink further.

23. •Correction for temperature:
For every 3 degree rise in urine temperature add 0.001 to the reading
of caliberation temperature. Subtract 0.001 from the reading for
every 3 degree below the caliberated temperature.
•Correction for abnormal solute concentration:
High SG in presence of glycosuria or proteinuria will not reflect true
kidney function(concentration ability) For nullifying the effect,
subtract 0.003 from the temperature corrected SG for every 1gm of
Protein/dl urine and 0.004 for every 1gm of glucose/dl urine.

24. 2. REFRACTOMETER METHOD
•Refractive index of total soluble solids:
Higher the concentration higher Refractive
Index and higer is the SG and vice-versa

25. 3.REAGENT STRIP METHOD:
• Concentration of ions in urine correlate with specific
gravity.

26. CHEMICAL EXAMINATION OF URINE
The routine urine analysis include chemical testing for
(1)Protein
(2)Glucose
(3)Ketone
(4)Occult blood
(5)Bile pigments
(6) Bile salts
(7)Urobilinogen.

27. DETERMINATION OF PROTEIN
• Small amount of low molecular weight protein is filtered at
glomerulus but prevent high molecular wt protein such as albumin
(mol wt 69000) and gamma globulin (mol wt. 1,80,000). Most protein
is reabsorbed in tubules.
•<150 mg protein/24 hrs is generally excreted which is not detected by
chemical method.
• Renal tubule secrete a mucoprotein called tamm –horsfall protein
which is normally excreted in urine.
.

28. RENAL PROTEINURIA
GLOMERULAR PROTEINURIA
• Glomerulonephritis
• Hypertension
• Lipoid nephrosis
TUBULAR PROTEINURIA:
• Pyelonephritis
• Renal tubular acidosis
• Cystinosis
• Interstitial nephritis
• Rejection of kidney allograft

29. PRERENAL CAUSE OF PROTEINURIA:
• Dehydration
• Intestinal obstruction
• Myocardial infarction
• Intra-abdominal tumor
POST RENAL CONDTIONS:
•Lesion of renal pelvis
•Bladder
•Prostate and urethra

30. TEST FOR PROTEINURIA:Sulfosalicylic acid precipitation test
•PRINCIPLE: Precipitation of Protein by chemical agent or
coagulation by heat.
•NOTE: Urine should be clear .If turbid, centrifuge it. Supernatant
tested for protein, Sediment for microscopic examination. If urine is
alkaline add few drops of glacial acetic acid make it slightly acidic.

31. REAGENT:
1. 3g/dl sulfosalicylic acid
2. Glacial acetic acid
PROCEDURE (A)
• Take 3 to 4 ml of centrifuged urine to small test tube
• Add 2 to 3 drop of sulfosalicyclic acid
• Observe turbidity after 5 minutes

32. OBSERVATIONS:
No formation of turbidity at upper portion of urine: Protein absent
Formation of turbidity : Protein present
Grade according to degree of turbidity +, ++ ,+++ and ++++

33. PROCEDURE (B) HEAT TEST

34. QUANTITATIVE ESIMATION OF PROTEINS:
Estimation of protein in 24 hrs urine sample
24 HR Urine creatinine is calculated.
Adult Male creatinine excretion is 14-26 mg/kg/24 hrs.
Adult Female :11-20mg/kg/24 hrs

P/C ratio
•Normal protein/creatine ratio:<0.2
•Low grade proteinuria: 0.2-1.0
•Moderate:1.0-3.5
•NEPHROTIC RANGE:>3.5

35. URINE REAGENT STRIP TEST FOR
PROTEIN
The principle is called as ‘protein error of indicators’ meaning that
one color appears if protein is present and another color if protein is
absent. Sensitivity is 5-10 mg/dl.

36. MICROALBUMINURIA: 30-300gm/24hr
• Microalbuminuria is the Earliest sign of renal damage in DM
(diabetic nephropathy).
• It indicates capillary permeability to albumin and denotes
microvascular disease.
• Also prevalent in hypertensive patient.
• Independent risk factor for cardiovascular disease.

37. Determination of glucose:
Presence of chemically detectable amount of glucose in urine is called
glycosuria.
Quantity depends on- blood sugar, GFR, degree of tublar reabsorption.
Normal renal threshold 150 – 170 mg/dl.

38. 1. HYPERGLYCEMIA: eg. Diabetes mellitus, hperthyroidism,
hyperadrenalism, hyperpituitarism, myocardial infarction,
cerebral hemorrhage, brain tumor, severe liver disease, and
disease of pancreas.
2.RENAL GLYCOSURIA: defect in tubular reabsorption
& subsequently lower renal threshold eg. Cystinosis,
heavy metal poisoning, fanconi syndrome ,depression of
renal function to 30% of normal or less like in acute RF

39. 3.Non pathological cause:
(a) pregnancy
(B) Stress and anxiety : increased epinephrine
(C) alimentary glycosuria: large carbohydrate diet following
low intake for week which decrease renal threshold.

40. COLOUR APPROX GLUCOSE g/dl
Green with precipitate 0.5
Brown precipitate 1.0
Yellow orange precipitate 1.5
Brick red >2.0
BENEDICT TEST: Done for estimation of reducing sugar.

42. URINE REAGENT STRIP TEST FOR
GLUCOSE
This test is more sensitive than Benedict’s qualitative
test and specific only for glucose. Other reducing agents
give negative reaction.

43. DETERMINATION OF KETONES
Inadequate carbohydrate in diet or defect in carbohydrate
metabolism, body metabolises fatty acids due to this ketone bodies
increase in blood.
1.Acetone
2.Acetoacetic acid
3.Β hydoxybutyric acid
Ketonuria is associated with diabetes
mellitus,anorexia,fasting,starvation,fever and prolonged vomiting

44. ROTHERA’S TEST
Nitroprusside used in the test react with acetone and acetoacetic acid
in presence of alkali(ammonium hydroxide) to produce a purple
coloured compound
Appearance of purple permangate
ring shows presence
ketone bodies in the urine

45. REAGENT STRIP TEST
It is a modification of nitroprusside test ,
it has sensitivity of 5-10mg/dl of acetoacetate.

46. DETERMINATION OF BILE PIGMENTS AND UROBILINOGEN:
PROCEDURE :
1.Place 3 to 4ml of urine in a centrifuged tube add equal amount of
10g/dl barium chloride, mix well.
2.Centrifuge at 1500 RPM for 10 minutes.
3. Place supernatant in another test tube for urobilinogen test.
4. Filter the remaining part on filter paper to obtain sediment for
bilirubin test.

47. FOUCHET’S TEST: On the sediments add 1 drop of fouchet
reagent, immediate development of blue green color around
drop indicate presence of bilirubin.
•EHRLICHS ALDEHYDE TEST: On the supernant add
ehrlichs reagent which react with urobilnogen to produce pink
colour. Intensity of colour is proportional to amount of
urobilinogen present.

48. Cherry red colour urobilinogen increased

49. DETERMINATION OF BILE SALTS
HAY’S SURFACE TENSION TEST
Bile salts when present lower the Surface Tension of urine.
When sulphur powder added on surface of urine, sulphur
particle sink to bottom of test tube.
In Normal urine sample sulphur particles float on surface of
urine.

50. DETERMINATION OF OCCULT BLOOD
Clinical significance
1.Hematuria
2.Renal disease: acute infection, glomerulonephritis TB, nephrotic syndrome, toxic damage,
malignant hypertension, renal calculi, trauma, tumors etc
3.Bleeding disorder-leukemia, thrombocytopenia, coagulation factor deficiency, sickle
disease or trait, scurvy etc.
4.Anticoagulant drugs
5. Haemoglobinuria
6. Myoglobinuria

51. PRINCIPLE:Peroxidase activity of haemoglobin decomposes hydrogen peroxide and liberated
Oxygen oxidises benzidine to form green blue colour complex.
PROCEDURE: Place pinch of benzidine powder and add 2 to 3 drop of glacial acetic acid in
test tube after that add 2ml of hydrogen peroxide solution now add 0.5 ml of urine and mix
well. Observe colour after 5 min.
OBSERVATION :Colour changes to Green or Blue :Occult blood present.
No Colour change : occult blood absent
BENZIDINE/ORHTOTOLUIDINE TEST

52. COLOR REPORT
FAINT GREEN TRACE
GREEN +
GREENISH
BLUE
++
BLUE +++
DEEP BLUE ++++

53. PORPHYRINS
PORPHYRIN : Intermediate in biosynthesis pathway of haem
TYPES OF POPHYRINS:
•
Coproporphyrin
•Uroporphyrin
•Porphobilinogen
•
aminolevulenic acid
Main site of porphyrin production is bone marrow.
Congenital deficiency of enzymes of haem biosynthesis pathway
causing porphyria in which liver produces excess of porphobilinogen
and δaminolevulinic acid.

54. Certain acquired conditions such as toxicity of heavy metals ,chemicals, acute
alcoholism and cirrhosis of liver and blood dyscrasia like leukemia, pernicious anemia,
hemolytic also increases the level of urine porphyrins.
DETERMINATION OF URINE PORPHOBILINOGEN
WATSON SCHWARTZ TEST:
Place 2.5 ml of urine and add equal qantity of modified ehrilch
reagent in test tube,then add 5ml of saturated sodium acetate and
mix with 5ml of chloroform, shake well and keep it for 5 min.
Chloroform carries urobilinogen to bottom.
If porphyrin present ,upper layer turn into red purple or deep red.

55. A.POSITIVE EHRLICH’S REACTION
B.WATSON SCHWARTZ TEST

56. MULTISTIX REAGENTN STRIP TEST
Reagent strip is the reliable frontline test for detection of a broad
range of conditions, from detecting urinary tract infections (UTI1
) to
diabetes and kidney disorders
A urine test strip or dipstick is a basic diagnostic tool used nowadays
to determine pathological changes in a patient's urine in
standard urinalysis.

58. THANKYOU

59. REFERENCES
•Fundamentals of urine and body fluid ananlysis by Nancy A. Brunzel
•Essentials of clinical pathology