Question

create a urinalysis lab with several samples. Please be sure to include the following: -Materials and methods -procedure -result -conclusion -data table

Answer 1

Examination of urine is important for diagnosis and assistance in the diagnosis of various diseases. Routine (complete) examination of urine is divided into four parts:

A. Adequacy of specimen

B. Physical/gross examination

C. Chemical examination

D. Microscopic examination

A. ADEQUACY OF SPECIMEN

The specimen should be properly collected in a clean container which should be properly labelled with name of the patient, age and sex, identity number with date and time of collection. It should not show signs of contamination. Specimen Collection For routine examination a clean glass tube is used; for bacteriologic examination a sterilized tube or bottle is required. A mid-stream sample is preferable i.e. first part of urine is discarded and mid-stream sample is collected. For 24 hours sample, collection of urine is started in the morning at 8 AM and subsequent samples are collected till next day 8 AM. Methods of Preservation of Urine Urine should be examined fresh or within one hour of voiding. But if it has to be delayed then following preservatives can be added to it which prevent its decomposition:

i. Refrigeration at 4°C.

ii. Toluene: Toluene is used 1 ml per 50 ml of urine. It acts by forming a surface layer and it preserves the chemical constituents of urine.

B. PHYSICAL EXAMINATION

Physical examination of urine consists of volume, colour, odour, reaction/pH and specific gravity.

Volume :Normally 700-2500 ml (average 1200 ml) of urine is passed in 24 hours and most of it is passed during day time.

i) Nocturia : Nocturia means when urine is passed in excess of 500 ml during night. This is a sign of early renal failure.

ii) Polyuria : Polyuria is when excess of urine is passed in 24 hours (> 2500 ml). Polyuria can be physiological due to excess water intake, may be seasonal (e.g. in winter), or can be pathological (e.g. in diabetes insipidus, diabetes mellitus).

iii) Oliguria: When less than 500 ml of urine is passed in 24 hours, it is termed as oliguria. It can be due to less intake of water, dehydration, renal ischaemia.

iv) Anuria: When there is almost complete suppression of urine (< 150 ml) in 24 hours. It can be due to renal stones, tumours, renal ischaemia.

Specific Gravity:This is the ratio of weight of 1 ml volume of urine to that of weight of 1 ml of distilled water. It depends upon the concentration of various particles/solutes in the urine. Specific gravity is used to measure the concentrating and diluting power of the kidneys. It can be measured by urinometer, refractometer or reagent strips.

1. Urinometer:Procedure Fill urinometer container 3/4th with urine. Insert urinometer into it so that it floats in urine without touching the wall and bottom of container read the graduation on the arm of urinometer at lower urinary meniscus. Add or substract 0.001 from the final reading for each 3°C above or below the calibration temperature respectively marked on the urinometer.

2. Refractometer: It measures the refractive index of urine. This procedure requires only a few drops of urine in contrast to urinometer where approximately 100 ml of urine is required.

3.Reagent Strip Method:

This method employs the use of chemical reagent strip.

Significance of Specific Gravity:The normal specific gravity of urine is 1.003 to 1.030.

Low specific gravity urine occurs in: i. Excess water intake ii. Diabetes inspidus

High specific gravity urine is seen in:

i. Dehydration ii.

Albuminuria

iii. Glycosuria

Fixed specific gravity (1.010) urine is seen in:

i. ADH deficiency

ii. Chronic nephritis

C. CHEMICAL EXAMINATION:Chemical constituents frequently tested in urine are: proteins, glucose, ketones, bile derivatives and blood. Tests for Proteinuria If urine is not clear, it should be filtered or centrifuged before testing. Urine may be tested for proteinuria by qualitative tests and quantitative methods. Qualitative Tests for Proteinuria

1. Heat and acetic acid test

2. Sulfosalicylic acid test

3. Heller’s test

4. Reagent strip method.

1. Heat and Acetic Acid Test ;

Heat causes coagulation of proteins. The procedure is as under:

Take a 5 ml test tube. Fill 2/3rd with urine. Acidify by adding 10% glacial acetic acid if urine is alkaline. Boil upper portion for 2 minutes (lower part acts as control). If precipitation or turbidity appears add a few drops of 10% acetic acid.

Interpretation:If turbidity or precipitation disappears on addition of acetic acid, it is due to phosphates; if it persists after addition of acetic acid then it is due to protein.

Depending upon amount of protein the results are interpreted as under :

No cloudiness = negative.

Cloudiness against dark background = traces (less than 0.1 g/dl).

Cloudiness without granularity = +(0.1 g/dl). Granular cloudiness=++(0.1-0.2 g/dl) Precipitation and flocculation = +++(0.2-0.4 g/dl). Thick solid precipitation = ++++ (0.5 g/dl).

2.Sulfosalicylic Acid Test :This is a very reliable test. The procedure is as under:

Make urine acidic by adding acetic acid. To 2 ml of urine add a few drops of20% sulfosalicylic acid.

Interpretation; appearance of turbidity which persists after heating indicates presence of proteins.

3. Heller’s Test:Take 2 ml of concentrated nitric acid in a test tube. Add urine drop by drop by the side of test tube.

Interpretation Appearance of white ring at the junction indicates presence of protein.

4. Reagent Strip Method:Bromophenol coated strip is dipped in urine. Change in colour of strip indicates presence of proteins in urine and is compared with the colour chart provided for semiquantitative grading.

Quantitative Estimation of Proteins in Urine

1. Esbach’s albuminometer method

2. Turbidimetric method.

1. Esbach’s albuminometer method:Fill the albuminometer with urine upto mark U. Add Esbach’s reagent (picric acid + citric acid) upto mark R. Stopper the tube, mix it and let it stand for 24 hours. Take the reading from the level of precipitation in the albuminometer tube and divide it by 10 to get the percentage of proteins.

Turbidimetric method:Take 1 ml of urine and 1 ml standard in two separate tubes. Add 4 ml of trichloroacetic acid to each tube. After 5 minutes take the reading with red filter (680 nm).

Causes of Proteinuria :20 Normally, there is a very scanty amount of protein in urine (< 150 mg/day). Heavy proteinuria (> 3 gm/day) occurs due to:

i. Nephrotic syndrome

ii. Renal vein thrombosis

iii. Diabetes mellitus

Mild proteinuria (< 1.0 gm/day) occurs in:

i.Hypertension

ii. Polycystic kidney

iii. Chronic pyelonephritis

iv. UTI

v. Fever.

Tests for Ketonuria:These are products of incomplete fat metabolism. The three ketone bodies excreted in urine are: acetoacetic acid (20%), acetone (2%), and β-hydroxybutyric acid (78%). Tests for Ketonuria

1. Rothera’s test

2. Gerhardt’s test

3. Reagent strip test

1. Rothera’s Test :

Principle :Ketone bodies(acetone and acetoacetic acid) combine with alkaline solution of sodium nitroprusside forming purple complex. Procedure:

Take 5 ml of urine in a test tube.

saturate it with solid ammonium sulphate salt.

Add a few crystals of sodium nitroprusside and shake.

Add liquor ammonia from the side of test tube.

Interpretation:Appearance of purple coloured ring at the junction indicates presence of ketone bodies.

Gerhardt’s Test:It is not a very sensitive test. Procedure: Take 5 ml of urine in a test tube.

Add 10% ferric chloride solution drop by drop. Filter it and add more ferric chloride. Interpretation: Brownish red colour indicates presence of ketone bodies.

Gerhardt’s Test: It is not a very sensitive test. Procedure :Take 5 ml of urine in a test tube.

Add 10% ferric chloride solution drop by drop. Filter it and add more ferric chloride. Interpretation:Brownish red colour indicates presence of ketone bodies.

Test for Bile Derivatives in Urine :Three bile derivatives excreted in urine are: urobilinogen, bile salts and bile pigments. While urobilinogen is is excreted in normal amount in urine.

Tests for Bile Salts:Bile salts excreted in urine are cholic acid and chenodeoxycholic acid. Tests for bile salts are Hay’s test and strip method.

1.Hay Test :

Principle:Biile salts if present in urine lower the surface tension of the urine. Procedure Fill a 50 or 100 ml beaker 2/3rd to 3/4th with urine. Sprinkle finely powdered sulphur powder interpretation:If bile salts are present in the urine then sulphur powder sinks, otherwise it floats.

Tests for Urobilinogen: Normally a small amount of urobilinogen is excreted in urine (4 mg/24 hr). The sample should always be collected in a dark coloured bottle as urobilinogen gets oxidised on exposure to light. Tests for urobilinogen in urine are Ehrlich’s test and reagent strip test.

1. Ehrlich’s Test:

Principle:Urobilinogen in urine combines with Ehrlich’s aldehyde reagent to give a red purple coloured compound.

Procedure :Take 10 ml of urine in a test tube. Add 1 ml of Ehrlich’s aldehyde reagent. Wait for 3-5 minutes.

Interpretation:Development of red purple colour indicates presence of urobilinogen. A positive test is subsequently done in dilutions; normally it is positive in upto 1:20 dilution.

2. Reagent Strip Test:These strips are coated with p-dimethyl-aminobenzaldehyde. When strip is dipped in urine, it turns reddish-brown if urobilinogen is present Significance Causes of increased urobilinogen in urine

i. Haemolytic jaundice and haemolytic anaemia Causes for absent urobilinogen in urine

ii. Obstructive jaundice Tests for Bilirubin (Bile Pigment) in Urine Bilirubin is breakdown product of haemoglobin. Normally no bilirubin is passed in urine.

Following tests are done for detection of bilirubin in urine: 1. Fouchet’s test 2. Foam test 3. Reagent strip test

1. Fouchet’s Test

Principle:Ferric chloride oxidises bilirubin to green biliverdin.

Procedure:Take 10 ml of urine in a test tube. Add 3-5 ml of 10% barium chloride. Filter through filter paper. To the precipitate on filter paper, add a few drops of Fouchet’s reagent (ferric chloride + trichloroacetic acid).

Interpretation:Development of green colour indicates bilirubin.

2. Foam Test: It is a non-specific test.

Procedure:Orthotoluidine Test Take 5/10 ml of urine in a test tube. Shake it vigorously. Interpretation:Presenceof yellow foam at the top indicates presence of biluribin.

3. Reagent Strip Test :

Principle: It is based on coupling reaction of bilirubin with diazonium salt with which strip is coated. Dip the strip in urine; if it changes to blue colour then bilirubin is present

. Causes of bilirubinuria

i) Obstructive jaundice

ii) Hepatocellular jaundice.

Tests for Blood in Urine:Tests for detection of blood in urine are as under :

1. Benzidine test

2. Orthotoluidine test

3. Reagent strip test

1. Benzidine Test

Procedure:Take 2 ml of urine in a test tube. Add 2 ml of saturated solution of benzidine with glacial acetic acid. Add 1 ml of H2O2 to it.

Interpretation:Appearance of blue colour indicates presence of blood. Benzidine is, however, carcinogenic and this test is not commonly used.

Orthotoludine test:

Procedure:Take 2 ml of urine in a test tube. Add a solution of 1 ml of orthotoluidine in glacial acetic acid. Add a few drops of H2O2.

Interpretation:Blue or green colour indicates presence of blood in urine.

3. Reagent Strip Test

The reagent strip is coated with orthotoluidine. Dip the strip in urine. If it changes to blue colour then blood is present Causes of blood in urine

i. Renal stones

ii. Renal tumours

iii. Polycystic kidney

iv. Bleeding disorders

v. Trauma.