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Introduction of Urine Examination

Urine is a simple type of specimen and it is the most common test specimen in the clinical laboratory. It is a liquid waste produced by our kidneys, a clear, transparent fluid that normally has an amber colour. Urine examination completes into various phases i.e. physical examination, chemical examination and microscopic examination. Physical examination of urine says about colour, odour, clarity, volume, and specific gravity while chemical examination of urine includes the identification of protein, blood cells, glucose, pH, bilirubin, urobilinogen, ketone bodies, nitrites, and leukocyte esterase, etc. Microscopic examination informs about cells urinary tract, blood cells, crystals, bacteria, parasites, fungus, etc.

Sample collection for Urine Examination

A First-morning sample is preferable though urine voided at any time may be examination Urine sample must be collected in a clean and dry container for routine tests. If the specimens are not collected properly, the laboratory finding may be unreliable. Fr culture a clean and sterile container should be provided instructing the patient to collect midstream clean catch urine.

Personal hygiene for Urine Examine

Before collection of urine: patients should wash the genital area in cases of urine culture.

• Urine specimens should not be collected from women during their menstrual period.

Collection methods for Urine Examination

Mis stream urine is required for all examinations when the patient starts urinating the first  5-10 ml should be discarded. The rest of the urine should then be collected in a suitable container according to the test.

Macroscopic  Urine Examination

First, note the appearance of the urine including colour transparency etc. Normal urine is straw coloured and clear. The colour and transparency may be altered in different pathological conditions i.e deep yellow in jaundice, turbid if plenty of pus cells are present.

• pH acidic/alkaline

Normal freshly passed urine is slightly acid, with a pH of around 6.0. In certain diseases, the pH of the urine may increase or decrease.

pH

Principle:

Coloured indicator papers are dipped in the urine. The colour changes according to the pH. The paper is then compared with a standard control chart giving the corresponding figure.

Materials:

• Slide
• Pasteur pipette
• Universal indicator paper/ litmus paper

The urine sample must be fresh.

Method:

• Place on the slides a piece of indicator paper. Let a few drops of urine fall onto the paper from the Pasteur pipettes.
• Pick the strip of paper up and compare the colour obtained with those shown on the standard chart.
• Read off the pH unit given for the colour most closely matched to the test strip.

Result

• Normal pH about 6.0 ( normal range from 5.0 to 7.0)
• Acid pH 4.5 – 5.5 (If persistent: some forms of diabetes, muscular fatigue, acidosis)
• Alkaline pH 7.8 -8.0 (infection of the urinary tract, vegetarian diet)

pH and crystalline deposits During Urine Examination

Determination of the pH of useful for the identification of crystalline deposits some crystals are deposited in acid urine only, others in alkaline urine only. For example:

• ACid Urien : Oxalates, urine acid
• Alkaline urine: phosphates, carbonates

Urinary deposits found during Urine Examination

Principle: Urine contains microscopic elements in suspension ( cells, crystals). These elements are collected by centrifuging and a drop of the deposit is examined between a slide and coverslip. as all these elements would sediment in the urine if left for a few hours, they are known as urinary deposits. Midstream urine should be examined as soon as possible after collection. In certain diseases of the urinary tract, the urine deposits are considerably altered, the following elements may be found.

The following may be found in urine deposits

• Red blood cells
• Leukocytes
• Parasitic eggs and larva
• T. Vaginslis
• Yeast cells
• Crystals
• Sperms

Materials

• Hand centrifuge, if no electricity
• Electric centrifuge
• Centrifuge tube
• Pasteur pipette
• Slide and coverslip

Preparation of the deposit

• Mix the urine gently
• pour immediately into a centrifuge tube until 3/4 full.
• Centrifuge as medium speed for 5 minutes.
• Pour off the supernatant urine by inverting the tube quickly without shaking.
• Shake the tube to resuspend the deposit.
• Place 1 drop into the slide and cover with a coverslip, number the slide with the patient’s number and examine under the microscope at once, first using the x 10 objective and x 40 with condenser lowered and the iris – diaphragm closed, to enable transparent objects to be seen.

Keynotes on Urine Examination

There are normally no red blood cells (RBCs) cells in the urine but white blood cells (WBCs) may be present in few numbers. Normally it should be 1-2  high per field (HPF.)

The presence of many leucocytes, especially if in usually indicates a urinary tract infection. It is usual to express RBCs,  WBCs and casts terms of HPF and it is important always to use the same method of expressing.

For Microscopic Urine  Examination

Place one drop of urinary deposits on a clean glass slide, cover with glass and examine first with 10x confirm with 40x objective.

Urine Examine microscopically

Do not confuse the red cells with yeast cells.

Yeast is of 5 – 12 um size.

Round or oval bodies in which budding may be seen. They are not soluble in acetic acid. Yeasts are occasionally present in urine containing glucose. So check that the urine is fresh or not.

Trichomonas vaginalis

SIze: 15 um ( 2 red cells)

Shape round, globular

Motility motile in fresh urine ( they whirl and turn)

Undulating membrane: on the side

Flagella: 4 flagella, more or less visible.

Spermatozoa during Urine Examination

• Occasionally found in the urine of males and in females immediately after sexual intercourse.
• Head: very small (5 µm).
• Flagellum: long and flexible (50 µm)
• Motility: motile in very fresh urine. otherwise nonmotile.

Epithelial cell in Urine Examination

• Squamous epithelial cells are large rectangular cells, the product of desquamation, the epithelial cells come from, the ureter and the vagina.

Bladder cells

• Large cells, often diamond-shaped, with a distinct nucleus.

Renal cellsRenal cells are small are the size of 1-2 leukocytes, very granular.

The nucleus is refractile and clearly visible. they are almost always present with protein.

Casts: Caster is cylindrical in shape and along, crossing almost the whole field when examined under the x 40 objects.

Types of casts encountered during Urine Examination-

• Hyaline casts in the urine examination

Transparent and slightly refractile, the ends rounded or tapered.

Granular casts Coarse and fine type

• Rather short casts filled with large granules, pale yellow in colour, with rounded ends. ( the granules from degenerate epithelial from the tubules of the kidney).

Fine granular casts in the urine examination

• The granules are smaller and do not fill the cast, do not confuse with hyaline casts, partly covered by amorphous phosphate crystals.

Blood casts in the urine examination

• Casts filled with more or less red blood cells, brownish in colour.

Pus casts: Casts filled with a move or less white blood cells.

Epithelial casts in the urine examination

• Casts filled with pale yellow epithelial cells. To make the cells more distinct, add a drop of 10%gm acetic acid solution to the deposit.

Fatty casts (rare)

• Very refractile yellowish casts, the edges indented and distinct.

Normal Crystalline deposits in Urine Examination

Crystals

Crystals have a regular geometric shape,

Calcium oxalate (acid urine)

Shape: like an envelope

size: about the size of 1-2 RBC

Urine acid (acid urine)

shape: varies (square, diamond-shaped, cubical or rose-shaped)

size: 30-150 µm, maybe very small size

colour yellow or brownish-red

Tripple phosphates ( neutral or alkaline urine)

shape: rectangular or like a fern leaf or star

colour: colourless, refractile.

Biochemical Tests in Urine Examination

Sugar tests in urine

Principle.

Glucose is a reducing substance. It reduced the blue copper sulphate of benedicts solution to a red cuprous oxide, which is insoluble.

Reagent

• Benedicts solution
• Tests tubes
• Boiling water bath
• Pipette. 1ml and 4 ml.

Method ( for urine sugar)

• Take a clean test tube and put 5 ml benedicts solution.
• Add 8 drops of urine mix well.
• Boil for 3-5 min into a boiling water bath or over a spirit lamp.
• Report the result as follows.

Colour: Result

Blue:  Negative

Green with yellow precipitate:   +

Yellow to dark green with precipitate: ++

Brown with precipitate:  +++

Orange to brick red with precipitate: ++++

Protein test in urine Examination

Principle

When sulphosalicylic acid is added to urine containing protein a white precipitate is formed. The urine must be clear if cloudy, use supernatant obtained after centrifugation.

Reagent

• Sulphosalicylic acide.
• Test tube
• Pipettes 1ml and 5ml

Method

• Take a clean test tube and about 5ml of urine.
• Put 1-2 drops of 30% sulphosalicylic acid into the urine.
• Compare with a tube containing urine alone; against a black ground.

Results (report the result as follows)-

Clear     –    Negative

Slightly cloudy: Trace

Cloudly:  +

Cloudly with small precipitate:  ++

Cloudly with moderate precipitate:  +++

Very cloudly with large precipitate: ++++

Urine protein (Heat and acetic acid method)

Reagent

5% acetic acid solution

Method

• Fill a test tube almost to the top with urine. Hold the test tube by the room and heat only the top part in a spirit lamp.
• After the urine boils, remove it from the spirit lamp, and add 2 or 3 drops of 3% acetic acid.
• If the urine is cloudy after adding the 5% acetic then the test for protein is positive.
• Somethings the urine becomes cloudy while heating but the cloudly ness goes away after adding 5% acetic acid. In this case, the test for protein is negative.

Urine sugar (strip method)

Principle

Glucose is oxidised by glucose oxidase to Gluconic acid and hydrogen peroxide. Hydrogen peroxide is broken down by peroxidase into nascent oxygen and water molecule.

Nascent oxygen reacts with a chromogen to give a shade of yellow to dark brown depending upon the amount of glucose present in urine.

A complete kit is available to buy commercially. A leaflet containing principle. Procedure limitation and interpretation is included inside it. It is absolutely necessary to follow the direction exactly.

Method

• Dip the strip into a fresh urine sample and withdraw immediately.
• Remove excess urine from the strip by touching the edge of the strip on the wall of the container.
• Lay the strip flat on the container facing the reagent area upward.
• Match the colour of the strip exactly after 30 seconds with the colour chart provided.
• Note the result according to the chart provided.

Bilirubin Test  in Urine Examination

Bilirubin is a coloured pigment that can be detected in urine in certain liver diseases, i.e acute hepatitis, cholelithiasis, obstructive jaundice, etc. A test for bilirubin should be performed when the dark colour of the urine indicates its possibility. Several methods are in use they are:

• Fouchet’s test
• Iodine test
• Gmelin’s nitric acid test
• Godfried test
• Hunter’s Diazo test

Fouchet’s test

This is a commonly used method sufficiently sensitive for clinical purposes.

Principle

Barium chloride reacts with the sulphate radicals in urine to form a precipitate of barium sulphate into which bilirubin has adhered. The Fouchet’s reagent will oxidise bilirubin to biliverdin which gives green colour.

Materials

Test tubes

Filter paper

Centrifuge tube or other suitable sized.

Procedure

• Acidify the urine if it is alkaline.
• Add 5ml of 10% barium chloride 10ml. of urine and mix well.
• Filter through Whatman no 1 filter paper.
• Spread the filter paper on another dry filter paper.
• Add a drop of Fouchet’s reagent to the precipitate.

Result: If bite pigment is present, a green colour develops. the intensity is proportional to the concentration of bile pigment, present in urine.

Iodine test

A few ml. of fresh urine is taken in a small tube and some alcoholic solution of iodine is layered into it.

Result:- A green ring junction of two fluids indicates the presence of bilirubin.

The method is not very sensitive.

Gmelein’s Nitric acid test

• Take a few ml. of concentrated nitric acid.
• Layer some urine over it.
• A violet or green-blue ring appears at the junction of two fluids is present Maximum colour develops at the end of 15 min.

Icto test (reagent table)

Composition of the tablet.

• P- nitrobenzene diazonium p- toluene sulphonate, salicylsulphonic acid and sodium hydrogen carbonate.

Principle

When urine is placed on a special mat, bilirubin is adsorbed on its surface. The slight effervescent poverty of the tablet partially disintegrate it and cause the reagent to wash onto the surface of the mat, where the bilirubin couper with the diazo compound is the presence of salicylsulphonic acid forming a bluish-purple compound.

Procedure

• Place five drops of urine on the asbestos-cellulose mat provide with the kit.
• Place a reagent table in the centre of the moistened area of the mat.
• Flow two drops of water over the tablet.
• Observe the colour of the amount around tabled for exactly 30 seconds.

Result:-

Negative: slightly red or pink colour around the tablet.

Positive: blue to purple around the tablet within 30 seconds.

Sensitivity of the test:

0.05-0.1 mg/dl.

Precaution:-

• The tablet is hygroscopic.
• Keep the bottle tightly closed when not in use.
• Protect the reagent tablet from direct sunlight.
• If the tablet is decolourised discard it.
• Test for positive and negative control when opening each batch of tablets.

Urobilinogen in Urine Examination

Urobilinogen is normally present in urine in a small amount.

Qualitative test for urobilinogen.

Principle

The test is based on Ehrlich’s aldehyde reaction in which urobilinogen forms a cherry colour with Ehrlich’s reagent.

Method

• To 10 ml of urine, add 1 ml of Ehrlich’s reagent.
• To 10 ml. of urine add 1 ml of 20% hydrochloric acid (HCl).
• Mix by inversion and stand for 3-5 minutes at room temperature.

Result:- Faint pink colour-  urobilinogen normal.

Cherryred- (Deep red colour) Urobilinoge increased (excess) = positive.

Strip method

Chemostrip is one of the strips which is impregnated with 4 merhoxybenzene diazonium tetrafluoroborate which couples with urobilinogen in an acid medium to form a red dye.

Ketone bodies Test in Urine Examination

Ketone bodies include acetoacetate or diacetoacetate, acetone and beta-hydroxybutyric acid. The usual methods applied are able mostly to detect acetoacetate and acetone. Normally there are no ketone bodies detectable by the usual methods in the laboratory. Ketone bodies are found in urine when there is increase fat metabolism i.e diabetes mellitus, starvation etc. Following methods are in common use to detect the ketone bodies.

1. Rother’s nitroprusside test
2. Nitroprusside tablet test
3. Reagent method

Rothera’s test:

This is the most common to detect acetone or acetoacetic acid in urine.

Principle

Acetoacetic acid or acetone, when treated with sodium nitroprusside in alkaline solution. produce a purple colour.

Reagents and materials

• Sodium nitroprusside
• Ammonium sulphate
• Concentrated ammonia solution
• Test tube rack, etc.

Procedure

• Take about 5ml of fresh urine in a test tube.
• Saturate the urine with ammonium sulphate.
• Add a small crystal of sodium from the side of the tube so that there become two layers of urine and ammonia solution.

Result: A purple coloured ring develops at the junction of the two-fluid if acetone is present.

Rothera’s table test:

Similar reagents to Rothera’s test are included in ‘Acetest. Place the drop of urine on the table and an example for a purple colour at the end of 30 seconds. The significance is similar to Rothera’s tets.

Method

• Dip the strip in fresh urine. Drain excess urine against the edge of the container.
• Lay the strip, on a surface.
• Read the strip at 10 to 30 seconds comparing it with the colour chart provided.

Sensitivity of the method:

This method detects about 0.4 mg/dl, values up to 1 mg/dl. are considered normal.

Precaution

• Urine should be fresh
• Strips are affected by the metabolites of drugs such as phenazopyridine and another compound such as a zogatrisin (false positive)

Bile salts in the urine

Normal urine contains a very small amount of bile salt which can not be detected from the usual method used in the laboratory.

Bile salts are present along with bile pigments in the urine in obstructive jaundice but they can be absent when pigments are still present during recovery.

Detection of bile salt in urine.

Hay’s (Sulphur) test in Urine examination

Principle: Bile salt lowers the surface tension which can be used for their detection.

Reagents: Flower of Sulphur

Method:

Sprinkle a little finely powdered flowers of sulphur into the surface of fresh clear urine in a test tube or small beaker.

Sulpher particles sink in presence of bile salt since bile salts reduce the surface tension of urine.

Sulphur particles remain on the surface of urine in the absence of bile salt. Showing negative results.

Bence Jone’s protein in urine examination

Bence Jone’s proteinuria is associated with multiple myeloma macroglobulinemia and malignant lymphoma.

Bence Jone’s protein precipitates at temperatures between 40-60 °C whereas other proteins precipitate between 70-70 °. On raising the temperature to boil, Bence Jone’s protein will redissolve but other proteins will not. On cooling to 60° C the Bence Jone’s protein re- precipitates.

Bence Jone’s protein gives a reaction with sulfosalicylic acid reaction but can be missed with the boiling test.

Test for Bence Jone’s protein

Harrison’s three tube test method

1. Filter urine and adjust reaction with 33% acetic acid until acid.
2. Place 5ml. of this urine into each of the three tubes.
3. To the three tubes, add 0,1 and 2 drops of 33% of acetic acid and mix ( to provide variation in the PH)
4. Place a thermometer inside the test tube and immerse the tubes in a beaker of cold water and heat slowly.
5. Carefully observe the temperature and any sign of precipitation.

It is advisable to store the water to maintain a uniform temperature.

Result:

When Bence Jone’s protein is present ay least one tube will go turbid at a temperature between 40-60°C  which disappears on raising the temperature to boil.

This method is not a very reliable method, the only satisfactory method of detecting Bence Jone’s protein is by Electrophoresis, the urine is concentrated about 5 times beforehand by using lyphogel.

Pregnancy Test in Urine examination

In pregnancy tests, we determine, qualitatively or quantitatively the human chorionic gonadotropin (hCG) produced by trophoblastic tissue. Clinical applications of ”Pregnancy test include confirmation of the clinical diagnosis of pregnancy early in the first trimester, identification of pregnant patients before ordering medications or radiologic examinations, diagnosis of ectopic pregnancy, evaluation of the threatened abortion and guidance of diagnosis and treatment of trophoblastic tumours.

Method of pregnancy tests

Five general methods are available to measure HCG activity

1. Bioassay
2. Agglutination immunoassay
3. Haemagglutination inhibition
4. Latex particle agglutination inhibition
5. Direct agglutination of latex particles.

Strip or card method

This is a rapid method for confirming pregnancy.

One of this method (Biostrip P) method utilises a solid phase, two-site immunometric assay in a combination of monoclonal and polyclonal antibodies to selectively detect elevated levels of hCG in urine with a high degree of sensitivity, In the test procedure urine is added to the tube with the aid of transfer pipette and Biostrip device is inserted into the test tube. If hCH is present in the specimen it will react with conjugate dye which binds to the antibody on the bio strip and generate a coloured line.

The strip or card method is very sensitive and given comparable results with ELISA. It can detect as low as 25 IU/ml of hCG in urine. The method is very simple, reliable and cost-effective.

This method can detect the pregnancy during the first day of missed period.

ELISA Technique for hCG detection.

This method is the most sensitive technique and is reliable if performed properly.

Principle:

The well coated with anti hCG is incubated are incubated with a urine sample and hCG present in the sample will combine with anti hCG- After washing an Enzyme-linked anti hCG is added and incubated. The enzyme-linked anti hCG will combine with the hCG- anti hCG complex in the wall. After washing specific substrate for the enzyme is added and the hydrolysis of the substrate is indicated by a suitable indicator.

ELISA technique is lengthy compared to other methods. It is routinely performed.