The document provides comprehensive information on urine analysis, including its composition, physical and chemical characteristics, and methods for collection and examination. It details the steps involved in routine urinalysis and highlights the significance of various constituents and their abnormalities, such as proteinuria, glucosuria, and ketonuria. The analysis procedure encompasses macroscopic, microscopic, and chemical tests to detect normal and pathological substances in urine.

1. Urine Diagnosis
Dr. V R
M.B.B.S., M.D.
Clinical Biochemistry

2. Routine Urine Analysis
Urine consists of:
(96%)
water
Inorganic:
Cl-
, Na, K.
trace amounts of:
sulfate, HCO3 etc.)
Urine:
• Is an ultrafiltrate of plasma from which glucose, amino acids, water and
other substances essential to body metabolism have been reabsorbed.
• Urine carries waste products and excess water out of the body.
(4%)
dissolved solids:
(2%)
Urea: (half)
(2%)
Other compounds
Organic:
creatinine
uric acid

4. Urine Analysis:
Routine Urinalysis (Routine-UA):
• It consists of a group of tests performed as part of physical
examination. It involves macroscopic and microscopic analysis.
Type of analysis:
• macroscopic analysis:
• microscopic examination: urine sediment is examined under microscope
to identify the components of the urinary sediments.
physical characteristics
chemical analysis

5. Steps in basic urine analysis
Three steps analysis:
First: physical characteristics of urine are noted and recorded.
Second: series of chemical tests is run.
Third: urine sediment is examined under microscope to identify the
components of sediments.
Factors affect on urine constituents:
dietary intake,
physical activity,
body metabolism,
endocrine function
others.

6. Urine Collection:
Sterile container
Types of urine specimens:
• type of specimen and collection procedure are determined by
physician and depend on the tests to be performed.
There are basically four types of urine specimens:
1. First morning specimen
2. Random urine specimen
3. Fractional collection
4. Timed collection
• Composition and concentration of urine changes during 24hr
• Urine conc. vary according: to water intake and physical activities.

7. Truly representative sample:
• Ist morning sample is preferred (particularly for protein
analysis) because they are more concentrated from
overnight retention in bladder.
• Mid stream sample: free of contamination, Free of debris or
vaginal secretions
• Time of analysis:
- must analyzed within 1h at room temp. or within 8hr at 2o
C- 8o
C
- If not assayed within these time limits, several changes will occur.
• sample should collected in a clean container.
• urine container must be sterile if the urine is to be cultured.
• For microscopic examination, the urine must be fresh.

8. I- Physical Characteristics:
• direct visual observation.
• Normal fresh urine: Color: pale or dark yellow-amber, clear.
• Vol:750 - 2000 ml/24hr.
• Physical examination involves:
1.Color
2.Transparency
3.Odour
4.Volume
5.pH
6.Specific gravity

9. Color:
• Many things affect urine color, including fluid balance, diet,
medicines, and diseases.
• Color intensity of urine correlates to concentration.
• Darker color means more concentrated sample.
•Amber yellow Urochrome (derivative of urobilin, produce
from bilirubin degradation, is pigment found in normal urine).
• Colorless due to reduced concentration.
• Silver or milky appearance Pus, bacteria or epithelial cells
• Reddish brown Blood (Hemoglobin), also phenolphthalein‐
containing laxatives, doxorubicin, and phenothiazines
• Yellow foam Bile or medications.
• Orange, green, blue or red medications. Rifampicin-Orange red
•Vitamin B supplements can turn urine bright yellow.

10. Transparency:
• Urine is normally clear. Bacteria, blood, sperm, crystals, or
mucus can make urine look cloudy.
• Is classified as clear or turbid.
• In normal urine: the main cause of cloudiness is crystals and
epithelial cells.
• In pathological urine: it is due to pus, blood and bacteria.
• A milky appearance may arise with lipiduria, chyluria,
and urinary tract infection with neutrophilia
• Degree of cloudiness depends on: pH and dissolved solids
 Turbidity: may be due to gross bacteriuria,
 Smoky appearance: is seen in hematouria.
 Thread-like cloudiness: is seen in sample full of mucus.

11. pH:
• pH measure acidicity or alkalinity (basic) of urine
• Normal urine pH: 4.5-8.
• Increased acidity in urine: due to diabetes or medications.
• Urine sample must be fresh (why?)
(on standing urine become alkaline as a result of ammonia liberation
due to urea decomposition).
• A urine pH of 4 is strongly acidic, 7 is neutral (neither acidic
nor alkaline), and 9 is strongly alkaline.
• Sometimes the pH of urine is affected by certain treatments.
For example, your doctor may instruct you how to keep your
urine either acidic or alkaline to prevent some types of
kidney stones from forming.: Phosphates will precipitate in an
alkaline urine, and uric acid will precipitate in an acidic urine

12. Specific Gravity (SG):
• measures the amount of substances dissolved in urine.
• also indicates how well kidneys are able to adjust amount of water
in urine.
• higher SG: more solid material is dissolved in urine
• When you drink a lot of fluid, your kidneys make urine with a high
amount of water in it which has a low specific gravity. When you do
not drink fluids, your kidneys make urine with a small amount of
water in it which has a high specific gravity.
• N = 1.010 and 1.030 (may range from 1.003 to 1.030)
• Specific gravity results above 1.010 can indicate mild dehydration.
The higher the number, the more dehydrated you may be.

13. Specific Gravity
Clinical Significance.
(1)Low specific gravity.
(a)Diabetes insipidus, a disease caused by impaired functioning of the antidiuretic hormone
(ADH), is the most obvious and severe example of the loss of effective concentrating ability.
This disease is characterized by large volumes of urine with low specific gravity. Specific
gravity in such cases usually ranges between 1.001 and 1.003.
(b) Low specific gravity may also occur in patients with glomerulonephritis, pyelonephritis,
and various renal anomalies. In these cases, the kidney has lost its ability to concentrate the
urine because of tubular damage.
(2) High specific gravity. Specific gravity is high in patients with adrenal insufficiency,
hepatic disease, and congestive cardiac failure. It is elevated whenever there has been
excessive loss of water, as with sweating, fever, vomiting, and diarrhea.
(3) Fixed specific gravity. Urine with a fixed low specific gravity (approx. 1.010) which
varies little from specimen to specimen is known as isosthenuric. This condition is indicative of
severe renal damage (renal failure) with disturbance of both the concentrating and diluting
abilities of the kidney.

14. Normal Chemical Constituents of Urine:
Organic: urea, uric acid, creatinine
Inorganic: Cl-
, PO4
-3
, HBO3, NH4, SO4
-2
1- Urea:
1ml urine + 3ml NaOCL (sodium hypochlorite) ==>Evolution of N2 gas.
2- Uric acid UA:
1ml urine + 0.5 ml 10% NaOH +1ml Folins reagent ===> Blue color.
3- Creatinine:
- 1ml urine + drops Picric acid + drops NaOH ====> red color ppt.
Note: if reaction is acidified with HCL, the color changes to yellow.

15. Chemical characterstics
• Protein. Protein is normally not found in the urine. Fever, hard exercise, pregnancy,
and some diseases, especially kidney disease, diabetic nephropathy may cause
protein to be in the urine.
• Glucose. Glucose is the type of sugar found in blood. Normally there is very little or
no glucose in urine. When the blood sugar level is very high, as in uncontrolled
diabetes, the sugar spills over into the urine. Glucose can also be found in urine
when the kidneys are damaged or diseased.
• Nitrites. Bacteria that cause a urinary tract infection (UTI) make enzyme that
changes urinary nitrates to nitrites.
• Leukocyte esterase (WBC esterase). Leukocyte esterase shows WBCs in the urine.
• Ketones. When fat is broken down for energy, the body makes substances called
ketones (or ketone bodies). These are passed in the urine. Large amounts of
ketones in the urine may mean a very serious condition, diabetic ketoacidosis, is
present. A diet low in sugars and starches (carbohydrates), starvation, or severe
vomiting may also cause ketones to be in the urine.

16. Identification of Pathological Physical and Chemical Urine
Constituents
Abnormal (Pathological) constituents of urine:
1- Macroscopic analysis:
 physical tests
 chemical tests
2- Microscopic analysis:
• Pathological urine constituents are substances which are not
usually present in urine such as glucose, protein, ketones,
RBCs, Hb, bilirubin…. etc.

17. How to detect abnormal constituents:
Urine strip:
• Strip is filter paper or plastic which has chemical
substance (reagent) coated on it on different pads.
• It gives color when react with substance in urine.
• The produced color is compared with chart color
visually or mechanically assessed.
Glucose
Bilirubin
Ketones
Specific Gravity
Blood
pH
Protein
Urobilinogen
Nitrite
Leukocyte

18. Results are reported as:
• In concentration (mg/dl)
• As small, moderate, or large
• Using the plus system (1+, 2+, 3+, 4+)
• As positive, negative, or normal
Automated Urine Testing
Machine
Urinalysis test strip

19. • This method is rapid, easy, give early indication and
qualitative.
• Therefore, usually there are other confirmatory tests:
(chemistry, microbiology and microscopic analysis).
• Reaction in strip is effected by time, to reduce timing errors
and to limit variations in color interpretation; automated
instrument is used to read the reaction color on each test pad.

20. Strip include the tests:
• Glucose
• Bilirubin
• Ketone
• Specific Gravity
• Blood
• Protein
• Urobilinogen
• Nitrite
• Leukocyte
• pH

21. Abnormal Urine Constituents include:
1- Proteinurea:
• is the presence of abnormal amount of protein in urine.
• Urine of healthy individual contains no protein due to:
 In normal physiology, small M.wt. proteins are reabsorbed by kidney
tubules (proximal tubule)
 large M.wt of protein so it can't pass through kidney tubule to urine.
unless kidney tubule has damage.
• The main protein in urine is albumin therefore,
proteinuria=albuminuria

22. Microalbuminuria:
• Is the presence of small amounts of albumin in urine.
• It is very important in detection of early stage of nephronpathy
and in diagnosis of DM complication (nephropathy).
• High protein in urine makes urine looks foamy.
• Associated with face or feet abnormal odema, due to
disturbance of liquid balance in body due to protein loss.

23. Detection:
• Qualitative test: using a reagent test strip.
• Quantitative test: depends on volume and time of urine
(protein conc. in urine may vary with time and volume)
• Most assays are performed on urine sample of 12-24h.
Reference value:
• Quantitative for 24-h urine:
Male:1-4 mg/dl Female: 3-10 mg/dl Child: 1-10mg/dl
• Qualitative reference value: Normal = Negative

24. 2- Glucosurea:
• is the presence of abnormal conc. of glucose in urine .
• Normally, glucose is reabsorbed by active transport in
proximal tubule and therefore doesn't appear in urine.
• If the blood glucose level exceeds the reabsorption capacity of
kidney tubules (renal threshold), glucose will appear in urine.
• Renal threshold of glucose: is around 180 mg/100 ml.

25. Semi-quantitative tests:
•Benedict’s Test
•Fehling’s Test
In Benedict’s test, Benedict’s solution is used as the reagent. Benedict’s reagent
is a combination of sodium carbonate, sodium citrate and copper sulphate
pentahydrate (CuSO4.5H2O).
Fehling’s test, Fehling’s solution-A and Fehling’s solution-B are used as the
reagents. Fehling’s solution-A is an aqueous solution of copper sulphate, having
blue colour, while Fehling’s solution-B is clear colourless aq solution of sodium
potassium tartrate.
On boiling the urine sample with the reagents, the copper sulphate (CuSO4)
present in the Benedict’s solution and Fehling’s solution is reduced by the
reducing agent, glucose (sugar), to form a coloured precipitate of cuprous
oxide

26. • Glucosuria indicates that glucose concentration in blood
exceeds this amount and the kidneys are unable to reabsorb it
efficiently.
Glucosuria occurs in DM, which characterized by:
• hyperglycemia,
• usually polyurea (increased volume of urine),
• high SG
• urine may be light in color.

27. 3- ketourea:
• is the presence of abnormal amount of ketone bodies in urine.
• Body normally uses carbohydrates as source of energy.
• If carbohydrate source depleted or there is defect in
carbohydrate metabolism, body use fat as a source of energy.
• Fat metabolism is occurred for certain time, at certain point,
fatty acid utilization occurs incompletely results in production
of intermediate substances (keton bodies).
• Three ketone bodies: acetone, acetoacetate, b-
hydroxybutayric acid
Oxidation
Fat Fatty Acids H2O+CO2+energy

28. • Normally ketone bodies are removed by liver.
• elevated levels of keton bodies in blood and urine cause acidosis
which leads to coma and death.
Ketourea is common in uncontrolled DM
because diabetic patient has high blood glucose but can't use by
cells, so lipids are used as source of energy.
• Ketourea present in:
 Disease
 Nutrition
 Vomiting for long time
• Results effected by: diet and drugs
Normal values: negative test result is normal.
Small: < 20 mg/dl Moderate: 30-40 mg/dl large > 80 mg/dl

29. 4- Haematourea:
• It is the presence of red blood cells (RBCs) in the urine.
• Can’t detected by the naked eye so detection by strip or
microscope as anucleated cells
• Positive result may be: normally: no pathological cause
abnormally: due to stones or tumers.
• Need other confirmatory test.

30. 5- Hemoglobinuria:
• Presence of heamoglobin in urine due to rupturing of RBCs
• This may occur in malaria, typhoid, yellow fever, hemolytic
jaundice and other diseases.

31. 7- Nitrite:
• used for screening for bacteria.
• Normal urine contain nitrate but not contain nitrites.
• In the presence of bacteria, the normally present nitrate in the
urine is reduced to nitrite.
• Positive test indicates presence of more than 10 organisms/ml.
reduction
nitrate nitrite "pink"
6- Bilirubin (Bile):
Result from hemoglobin breakdown
Elevated in hepatitis and jaundice (biliary duct obstruction).

32. 8- Urine leucocytes:
• This test detects any microbial infection in the body.
• Depends on esterase method:
+ve result: means more than 5 leucocytes/hpf. (high power field)
• If urine stand long time leucocytes lysis and more intense
reaction occur.
• False positives: occurs with vaginal contamination, presence
of glucose, albumin, ascorbic acid
• False negative: large amounts of oxalic acid can inhibit the
reaction.
Esterase + Ester 3-0H-5-phenyl pyrrole diazonium salt
pink -purple color
neutrophils reagent
strip