Useful Notes on the Procedure and Techniques of “Urine Analysis”

All urine samples become cloudy or turbid upon standing and so freshly passed urine should be examined. First morning specimen after getting up from bed is best for routine examination. Post-prandial specimen is collected one and half to three hours after principal meal.

Collection and Preservation:

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Containers used for collection of urine should be chemically clear. Urine for bacteriological examination should be collected aseptically, into sterile containers without preservation. Mid­stream of urine is preferred as it contains very few contami­nating bacteria.

Urine may be preserved for quantitative examination in a refrigerator or a small piece of camphor or thymol is added. Other preservatives include 2% toluene and formaline. For microscopic examination one to two drops of formaline (for 30 ml of urine) can be used.

Physical Examination:

Physical examination includes—quantity, colour, odour, appearance, reaction and specific gravity.

Quantity:

Urinary output of a healthy adult male is 1500 ml/day. However, variations may occur. Women and children excrete less urine. A greater amount of urine is voided during day than at night.

a. Daily output more than 2000 ml is known as polyuria.

Causes of Polyuria:

1. Excessive fluid intake particularly in cold weather.

2. Nervousness, excitement.

3. Diabetes mellitus and insipidus.

4. Hyperpyrathyroidism.

5. Renal failure.

b. Daily output less than 500 ml is known as oliguria.

Causes of Oliguria:

1. Dehydration.

2. Cardiac decompensation.

3. Pyrexia with poor fluid intake.

4. Acute glomerulonephritis.

c. Complete absence of urine formation is known as anuria. The different physiological conditions lead to anuria.

Colour:

Normal urine is pale yellow coloured. Following abnormalities in different conditions are observed.

Odour:

Normal urine has aromatic odour, and on standing it is ammonical, due to ammonia released by the activity of bacteria.

Pathologically:

a. Fruity or sweetish—Ketone bodies

b. Putrid—H2 S liberation, pus cells

c. Faecal—Due to contamination with faeces or coliforms.

d. Ammonical—Due to bacterial action.

Appearance:

Normal urine is clear. It may develop slight turbidity on standing. These clouds settle at the bottom of the container, which consists of pus cells, RBCs, epithelial cells and mucus. Bacteria in large number cause uniform cloudiness; Turbidity is due to presence of urates, phosphates, haematuria and chemicals.

Reaction or pH:

Normal urine is slightly acidic with pH 6 to 6.8. After full meal, the reaction may be alkaline or amphoteric. Reaction is measured by litmus paper or by nitrazine paper. It turns blue in alkaline and red in acidic urine. Nitrazine paper develops certain colours, which are then matched with standard colours.

Specific Gravity:

The specific gravity varies inversely with quantity of urine. Normal average is 1.010 to 1.025. It is determined by urinometer or refractometer as:

i. Take urine in a cylinder, upto nearly full; remove the froth of bubbles by filter paper. Float the urinometer, so that it does not touch the bottom or sides. Take the reading from lower meniscus.

ii. If the urine is insufficient, dilute it with an equal volume of distilled water, mix and take the specific gravity. The last two figures of the reading are multiplied by 2.

Example:

If the reading is 1.008, the specific gravity is,

08 ? 2 = 16

i.e. = 1.016.

Albumin increases the specific gravity. In such cases, a correction by deducting 0.001 for every 1% of albumin in urine should be made.

a. High specific gravity is seen in excess sweating, glycosuria, acute nephritis, albuminuria and all causes of oliguria.

b. Low specific gravity is seen in (less than 1.001) excessive water intake, chronic nephritis, diabetes insipidus, etc.

Chemical Examination:

It includes test for albumin, glucose, ketone bodies, bile salts, bile pigments, urobilinogen, etc.

Albumin:

Normal urine may contain trace, i.e. upto 2 to 3 mgm/100 ml of urine.

Albuminuria means presence of albumin and globulin in the urine. Following methods can detect albumin in urine.

a. Heat and Acid Test:

Make urine slightly acidic by adding few drops of 10% acetic acid. Fill 3/ 4th of a test tube with clear and fresh urine, filter, if necessary, and boil the upper part.

Turbidity indicates protein as phosphates. Add a few drops of 10% acetic acid, if the turbidity disappears, it was caused by phosphates and a persistence or increasing cloudiness indicates albumin in urine.

False negative results may be obtained if the reaction of urine is alkaline or it has a low specific gravity.

If entire specimen of urine coagulates, the albumin usually amount 2 to 3%.

b. Sulphosalicylic Acid Test:

The reagent contains sodium sulphate (750 gms) and sulphosalicylic acid 50 gms in 1000 ml of distilled water.

Mix equal quantities of urine and reagent gently by the side of the tube. A white cloud indicates presence of protein. Sulphosalicylic acid precipitates urinary protease, polypetides and Bence Jones proteins. A heavy precipitate to solid coagulation suggest 0.5% or more protein.

Causes of Albuminuria:

Prolonged expoxure to cold, pregnancy, severe muscular exertion, drugs and chemical poisoning, nephritis, tuberculosis or carcinoma of kidney, urethritis, prostatitis, etc.

Glucose:

Normal urine does not contain glucose. Glucouria means presence of glucose in urine. Following methods can detect glucose in urine.

Test for Glucose:

(For reducing substances like fructose, lactose, galactose, and pentose).

a. Benedict’s Test (Qualitative):

Take 5 ml. of Benedicts’ qualitative reagent in test tube and add 0.5 ml. of urine. Boil the content of tube. Let it stand on the rack for 5 to 10 min. The appearance of a yellow or red deposits indicates the presence of reducing substances, i.e. sugar. Cupric sulphate is reduced to cuprous oxide by boiling with reducing agents.

Report:

A slight green colour, light turbidity or a bluish white ppt or no change is reported as negative. A greenish colour with a little yellow deposit is reported as a trace (+), green yellowish (++) orange (+++) and brick red (++++).

Nowadays, paper strips are available commercially which are dipped in urine as directed and the colour produced is matched against the colour chart supplied.

Causes of Glycosuria:

Diabetes mellitus, non-diabetic glycosuria includes emotional disturbances, hyperthyroidism, pregnancy, after ingestion of considerable corbohydrates, either anaesthesia, in some infections, like pneumococcal pneumonia, etc.

Ketone Bodies:

Ketone bodies comprise of acetone, acetoacetic acid and (3 hydroxy butyric acid. Ketone bodies are products of incomplete fat metabolism and their presence is indicative of acidosis. Ketoneuria is commonly seen in uncontrolled diabetes mellitus, vomiting and diarrhoea. It is also present in vomiting of pregnancy and in hunger strikers.

Following methods can detect ketone bodies in urine.

Rothera’s Test:

Saturate 10 ml urine with Rothera’s mixture consisting of 99 parts of ammonium sulphate and 1 part of sodium nitropruside. Add slowly about 2 ml of strong ammonia. Allow it to stand for 5 min. A purple colour indicates presence of ketone bodies.

Nowadays acetest tablets are available for the detection of ketone bodies. In positive cases, addition of one drop of urine on this tablet results in the violet colour formation within 30 sec.

Presence of ketone bodies in urine is known as ketonuria. The causes include uncontrolled diabetes mellitus, starvation, dehydration and malnutrition, vomiting and diarrhoea, etc.

Bile Salts (Hay’s test):

Bile salts consist of glycocholic acid and taurocholic acid. They lower the surface tension of the fluid and thus cause sulphur particles to sink.

Take about 3 to 5 inch column of urine in a small beaker or in a test tube. Sprinkle finely powered dry sulphur over the surface from a height of about half inch. If bile salts are present, the sulphur powder will sink at bottom.

The presence of bile salts indicates obstructive jaundice.

Bile Pigments:

It is present in urine in obstructive jaundice and hepatocellular jaundice. The detection test includes:

Fouchets Test:

This is the most sensitive test. If the urine is alkaline or neutral, acidify it with few drops of (2%) acetic acid. To about 10 ml of acidic urine add about 5 ml or 10% barium chloride solution. Mix well and filter.

To the residue on the filter paper add a drop of Fouchets reagent. A green or blue color indicates pres­ence of bile pigments, i.e. biliverdin and bilirubin respectively.

Urobilinogen:

If the urine sample contains bile pigment it should be removed by addition of 1 part of 10% aqueous solution of calcium chloride to 4 parts of urine and filtering it. To 10 ml of fresh urine add 1 ml of Ehrlich Aldehyde Regent. Allow it to stand for 3 min. If red/cherry colour is obtained, it indicates presence of urobilinogen in urine.

Occult Blood:

Centrifuged urine deposit is the best to find the RBCs in the urine. Presence of more than one RBC per high power field (hpf) should be regarded as abnormal.

Microscopic Examination:

For microscopical examination a fresh concentrated specimen, preferably over night sample with restricted fluid intake, should be examined for cells, casts and crystals. If the urine is diluted or contamined, cells and casts dissolve very quickly.

Cells (Figs 22.2A and B):

i. RBCs: More than one per high power field is abnormal.

ii. WBCs: More than one per high power field is abnormal.

iii. Epithelial Cells: Squamous epithelial cells which may be excreted in moderate number are of no pathological importance.

All other cells should be regarded as pathological.

Figs 22.22A and B: Cells observed in urine

A

1. Transitional epithelial cells

2. Caudate epithelial cells

3. Renal epithelial cells

4. Renal epithelial cells with fatty degeneration

B

1. Pus cells in alkaline urine

2. Pus cells in acidic urine

3. Renal epithelial cells in acidic urine

4. Fresh RBCs

5. RBCs in urine with high specific gravity

Casts:

Casts have paralleled edges, ends, round or broken. They may be straight or curved, long or short. The finding of cast is very important. Its presence is usually indicated in some form of kidney disorder. Following casts can be seen in different abnormal cases:

i. Hyaline casts: It is colourless, homogenous, semi- transparent and cylindrical in shape. More than 1 per low power field is abnormal.

ii. Granular casts: These are hyaline casts, filled with granules.

iii. Waxy casts: These are more opaque than hyaline casts. It is usually seen in advanced chronic nephritis and amyloid disease.

iv. Epithelial casts: These contain epithelial cells in it.

v. Fatty casts: These contain numerous fat droplets.

vi. Pus casts: These are composed of pus cells.

vii. Blood casts: These contain RBCs in it.

Crystal:

In acidic urine, uric acid and urates are found in different forms, usually coloured. The crystals includes (Figs 22.5 to 22.7):

i. Oxalate: They are in the form of envelopes and sometimes can be seen as dumb-bells or biscuit shaped, i.e. calcium oxalate.

ii. Cystine: They are colourless and hexagonal. These are soluble in alkalis. Their presence is pathogenic

iii. Leucine and Tyrosine: These are rare crystals, associated with acute yellow atrophy of the liver and acute phosphorus poisoning. Usually, these crystals appear together. Leucine crystals are glistening; yellow spheroids and tyrosine crystals are fine silky needles.

In alkaline urine, following crystals are found.

i. Amorphous Calcium and Magnesium Phosphates: It is the commonest urinary sediments in the alkaline urine. It has no significance. It looks like small colourless granules.

ii. Ammonium, Magnesium (Triple) Phosphate Crystals: It appears as incomplete, irregular and colourless prisms, which vary in size and shapes.

iii. Magnesium Phosphates: These are elongated rhomboid flakes, large and refractive. Some are with rough edges and surfaces.

iv. Calcium Carbonate: Sometimes this is found in alkaline urine. It is a dumb-bell shaped, often found in pairs.

Parasites:

Urinany parasites are very few, including ova of schistosoma, microfilaria, trichomonas vaginalis and echinococcus.

Miscellaneous:

Miscellaneous urinary deposits includes—spermatozoa, urethral filaments, tissue debrits, mucus, bacteria, hair, dust, cotton wool, etc.