Routine examination of urine

ROUTINE URINE ANALYSIS

The routine analysis of urine includes the physical, chemical and microscopic observation.

Physical Examination of Urine

1. volume: Normal range is 1200-2000 ml/day. Increased quantity of urine is called Polyuria. Decreased quantity is called Aneuria.

2. Specific Gravity: The specific gravity of urine is the measure of the relative amount of Solution. Normal range is 1.005 - 1.025. Specific gravity is determined by  instrument is called Urinometer. A cylinder is filled with 100 ml of urine and the urinometer placed in it. It floats and after it becomes still,the reading is taken.

3. Odour: Normal fresh urine has a faint aromatic odour but on standing it decomposes and gives ammonical odour.

4. Turbidity: Fresh and normal urine is clear. Normal urine may appear cloudy due to amorphous phosphates in alkaline urine or amorphous urates are present in acidic . Amorphous phospate form white precipitate which dissolves when acid is added. Amorphous urates form pink coloured precipitate which dissolves when the specimen is heated.Uine may appear cloudy or turbid due to the presence of leukocytes,epithelial cells and bacteria.

5. Colour: The colour of normal urine may vary from pale yellow to dark amber.depending on the concentration of the pigments urochrome. Very pale or colorless urine can result from high fluid consumption, diuretic drugs, natural diuretics such as alcohol and coffee and also clinical condition such as diabetes insipidus and diabetes mellitus.

6. pH:  A high protein intake and ingestion of acidic fruits produces acidic urine. Diet that is high in vegitables and citrus fruits causes an alkaline urine .Normal range of fresh normal urine is 4.6 -7.0 (average 6.0)

Chemical examination of urine

Chemical examination of urine includes Protein, Glucose, Ketone bodies, Occult blood, 

Bile pigments, Bile salt and Urobilinogen. 

1. Determination of Protein : There are two methods 1) Sulfosalicylic acid method 2) Heat and acetic acid method. Sulfosalicylic acid method: Transfer 3 to 4 ml of centrifuged urine to a small test tube. Add 2 to 3 drops of sulfosalicylic acid on the top of the specimen. Observe for turbidity after 5 minutes. After 5 minutes if turbidity is appear at the upper portion it is due to protein present in the urine. 

Heat and acetic acid test : Place 5 to 10 ml of clear urine in a test tube . Boil the upper portion over a flame. If turbidity develops, add 1 to 2 drops of glacial acetic acid.If the turbidity is due to phosphate precipitation, it will clear.Reboil the specimen.Observe after reboiling the specimen. Turbidity indicates the presence of protein.Grade the degree of turbidity as Fine trace,trace, +,++,+++,and++++.

Clinical significance of Protein: Increased level of protein in the urine is called proteinuria. There are two main mechanisms by which proteinuria can occur (1)  Glomerular damage (2) Defect in the reabsorption process of the tubules. Some of the conditions associated with glomerular proteinuria are glomerulonephritis, hypertension and lipoid nephrosis. Reabsorption related conditions are pyelonephritis, renal tubular acidosis,cystinosis and interstitial nephritis.

2. Determination of Glucose : Determination of glucose by Benedict's qualitative test.

Procedure: Pipette 5.0 ml of Benedict's reagent in test tube. By using pasteur pipette, add eight drops of urine. Heat carefully on the flame of a gas burner (or spirit lamp) or place in a boiling water for 5 to 10 minutes. Cool under tap water or by placing in a beaker containing tap water.

Clinical significance of Glucose : The presence of chemically detectable amount of glucose in urine is called Glycosuria (or glucosuria). It is mainly due to Hyperglycemia.

3. Determination of Ketones : Ketone bodies also called Acetone bodies. Chemically there are aceto acetic acid, β hydroxy acetic acid and β hydroxy butyric acid. Kertosis is type of condition in which the concentration of the above three increases in blood and urine. Acetone bodies are detected by Rothera's method. Take two test tubes and label them as T and C respectively. Add pich of Rothera's powder mixture in both test tubes. In the tube labeled as T, add one drop of urine. In the tube labeled as C, add one drop of distilled water. Observe colour of the reaction mixture after five minutes. If violet colour appear, it is due to the presence of ketone bodies in the urine sample.

Clinical significance of Ketone bodies : Detection of ketonuria in a patient with diabetese mellitus is of great significance since a change in insulin dosage or other management is often indicated. Ketonuria also accompanies the other condtions such as anorexia, fasting starvation, fever and prolonged vomiting.

4. Determination of Bile salts : Bile salts when present in urine lowers the surface tension. Five ml of urine is taken in a test tube. Add pich of sulphur powder and keep it for ten minutes. A positive reaction is observed, when the sulphur sinks to the bottom and a negative reaction when the sulphur remains floating.

5. Determination of Bile pigments and Urobilinogen : The constituents or derivatives of bile that may be present in the urine are bile pigments. Place 3 to 4 ml of urine in a centrifuge tube (about 1/3 full) by using a Pasteur pipette. Add equal amount of 10 g/dl barium chloride, mix well. Centrifuge at 1500 RPM for 10 minutes. (or Filter by using Whatman No.1 filter paper). Place supernatant in another test tube for urobilinogen test. Add one to two drops of Fouchet's reagent to the sediment (or to the precipitate on a filter paper). Add about 0.5 ml of Ehrlich reagent to the supernatant. A green or bluish green colour in the sediment indicate the presence of bile pigment. Supernatent colour change to cherry red colour, it is due to the incresed level of urobilinogen in the urine.

Clinical significance of Bile pigments,Bile salts and Urobilinogen : Determination of Bile pigments, Bile salts and Urobilinogen is useful in the diagnosis of jaundice.

6. Detection of blood cells (Occult Blood Test) : Blood present in the urine may be in the form of either intact cells , hematuria or haemoglobinurea 

Microscopic examination of urine

This is the most important procedures in urine analysis. T

he specimen should be collected in a sterile container.The microscopic elements present in urine are collected in the form of deposit by centrifugation. A small drop of the sediment is examined by making a cover slip preparation under microscope. Take 5 ml of urine in a test tube . Centrifuge for five minutes at 2500 RPM . Discard supernatant . Resuspend the deposit by shaking the tube. Place one drop of the deposit on a glass slide. Put cover slip and observe under high power objective. 

Leukocytes : These are degenarated leukocytes. Normal urine contain 2 - 3 pus cells/hpf. These are mostly neutrophils. The addition of 2% acetic acid makes the nucleus visible. Approximate diameter 10 - 12 μm

Epithelial cells : These cells may orginate from any site in the genito urinary tract from the proximal convoluted tubule to the urethra or from vagina. Normal urine contain 3 to 5 epithelial cells/hpf. Three types of epithelial cells may be in urine, tubular, transitional and sqamous. 

Erythrocytes : In fresh urine these cells have a normal, pale or yellow appearance.They do not contain nuclei. They appear smooth biconcave disks.In dilute urine the red cell swell up and lyse. Lysed cells appear as colorless circles. In hypertonic urine the red cell crenate.

Casts : Urinary casts are formed in the lumen of the tubules of the kidney. The renal tubules secreat a mucoprotein called Tamm-Horsfall protein which is believed to form the basic matrix of all castsl. a) Granular casts : These always indicate significant renal disease. The casts are present due to the degenaration of cellular casts or due to direct aggregation of serum protein in a Tamm-Horsfall mucoprotein matrix. b) Hyalin casts: They are colourless, homogeneous, transparent and with rounded ends. c) Red cell casts : The cast may contain only a few RBCs in protein matrix or there may be many cells packed close together with no visible matrix. These always indicate significant renal desease. d) White cell casts : White cells that appear in casts are polymorphonuclear neutrophils.Cells may be tightly packed together. These casts may be present in renal infection and in noninfectious inflammation. They can be seen in acute pyelonephritis and in glomerular disease. e) Epithelial cell casts : These cast may be arranged haphazardly and very in size and shape. These casts present in severe chronic renal disease. f) Waxy casts : These cast have very high refractive index. These are yellow grey or colorless and have a homogeneous appearence. g) Fatty casts : Fatty casts are formed by incorporated free fat droplets or oval fat bodies.These are seen in nephrotic syndrome, chronic glomerulonephritis and toxic renal poisoning.

Crystals : Cristals present in acid urine a) Uric acid cristal : Shape of uric acid crystal is diamond rhombic or rosette form. These are stained with urinary pigments as yellow or red brown. Presence of uric acid crystals can be normal occurence. These crystal can also be present in gout, chronic nephritis and acute febrile conditions. b) Calcium oxalate crystals : These are colorless and octahedral or envelop shaped. These crystals present in various clinical conditions such as oxalate calculi,diabetes mellitus and liver disease. c) Cystine crystals : These are colorless,refractile,hexagonal plates with equal or unequal sides. They occur in patients with either congenital cystinosis or congenital cystinuria. They can form calculi. d) Tyrosine and leucine in the form of needle and spheroids may be present in urine with acute yellow, atrophy of the liver. e) Cholesterol plates with their characteristic missing corners may be observed in Nephrotic condition. f) Sulfa crystals also present when sulfa drugs are administered.They may be clear or brown in colour. Cristals found in alkaline urine a) Calcium phosphate : These crystals are long, thin and colorless. The appearance is like prisms with one pointed end.They may also form calculi. b) Tripple phosphates : The cristals are colorless prisms with three to six sides and frequently with oblique end. These crystals found in chronic cystitis and enlarged prostate.

Bacteria : Freshly voided normal urine is genarally free of bacteria. Presence large number of bacteria with many pus cells indicates urinary tract infection.