Urinalysis and Renal Function Tests



Urinalysis and Renal Function Tests

Collection of Urine

Many urine estimations are carried out on timed specimens ( 24hrs ) . The accuracy of the result depends largely on that of urine collection . Therefore , more care should be taken to ensure that the collection of urine for 24 hours is done properly . For example when a 24 hours collection is required between 10 AM on Monday and 10 AM on Tuesday , the following procedure should be adapted. On Monday 10 AM, empty bladder completely and discard the specimen. Then collect all the urine passed in bottle containing the preservative till 10 AM on Tuesday . The bladder is emptied at 10AM on Tuesday and this is also added to the above pooled urine .

Preservation for Urine

A preservation must be added to the urine to prevent bacteria growth and destruction of the substance being estimated .

Hydrochloric acid

Acidification of the specimen is very satisfactory . 10cc of conc. HCl is adequate for 24 hour specimen . This is suitable for the determination of urea , ammonia , total nitrogen , calcium and phosphorus .Toluene is a commonly used preservation . It is convenient for sodium , potassium , uric acid and protein analysis . This only prevent further surface contamination with bacteria . Thymol is also used as a preservative ( a few crystals or 5 ml of 10% solution ) . This is a satisfactory preservative for a wide range of substances . However , it cannot be used for the estimation of 17-oxosteroidsusing Zimmermann reaction .

Composition of Urine

Colour

Normal urine is clear and straw coloured . The colour is due to the pigment urochrome . The colour is lighter when large amount of water is consumed and darker in fevers and after excessive perspiration . In jaundice , the urine is yellow due to excretion of bile pigments . In alkaptonuria and methaemoglobinaaemia urine is dark brown in colour .

Odour

Freshly voided urine has a mild aromatic odour . It develops ammoniacal odour on standing , due to the formation of ammonia by bacterial decomposition of urea . Urine in ketosis emits the smell of acetone .

Volume

The daily urinary output varies widely and dependents on the fluid intake and environmental conditions. The volume excreted varies between 1000-2000 ml . Polyuria or increase in the volume of urine is seen in diabetes mellitus . A fall in the urinary output is termed as oliguria which is found in renal failure , some diseases of the heart feand lungs , fevers and diarrhea . Anuria , a complete cessation of urine output is seen in the terminal stages of renal failure .

Appearance

Normal urine is ordinarily clear and transparent when freshly voided . It may become cloudy on standing because of the precipitation of phosphates . Urine may be turbid when RBCs or pus cells are present .

Specific Gravity

Specific gravity varies from 1.010 to 1.030 depending upon water and food intake . It is determined in clinical practice by means of a urinometer . It consists of a weighted cylinder which floats in urine and a stem caliberated in degrees of specific gravity usually from 1.000 to 1.060 . The depth to which the urinometer sinks depends on the density of the urine . The instrument is usually caliberated for use at 15[pic] .

Procedure

1.Fill the cylinder with urine without producing bubbles.

2. Float the urinometer so that it does not touch the sides.

3. Make the reading from the bottom meniscus .

Temperature Correction

The urinometer is usually caliberated for use at 15[pic] and specific gravity should be corrected to room temperature by adding .001 for every 3 [pic] above the temperature at which the urinometer is caliberated (15[pic] ) .

Room temperature = 30[pic]

Specific gravity read = 1.009

Temperature correction = 1.009 + 5 x .001

= 1.014

Total Solids

Under normal conditions , the total solids present is about 1500 ml of urine varies 60 to 70 gm . The output of urinary solids is influenced by the intake of fluid diet. The total solids per litre of urine is obtained by multiplying the last two digits of the specific gravity by a factor of 2.6 which is called the Longs Coefficient . If the specific is 1.020 , the total solids would be 52 g / litre .

Reaction

Normally urine is acidic (pH=6) in reaction due to monobasic salts of phosphoric acid plus small amounts of organic acids . The acidity of urine is influenced by diet , fluid intake and also by various drugs . Meat diet will increase acidity whereas fruit decreases . Fasting and starvation in which the body proteins are metabolized also tend to increase titrable acidity . Urine on standing turns alkaline due to the formation of ammonia by the bacterial decomposition of urea .

Constituents of Normal Urine

Normal urine is composed of : 1. Water , 2. Inorganic salts , 3. Organic compounds .

Major Inorganic Ions

Anions = Cl_ , PO4---- , SO4----

Cations = Na+ , K+ , Ca++ , Mg++ , [NH4]+

Organic Constituents

NPN Nonprotein nitrogen is nitrogen in the blood that is not a ,acid , creatine , creatinine and polypetitides . NPN substances in urine are urea , creatine , creatinine , uric acid , amino acids , allantion and hippuric acid .

Urea About 25 to 30 gm of urea is excreted daily in urine . As urea is the end product of protein metabolism , urea excretion in urine is an index of protein intake in diet . Urea in urine is increased in exaggerated protein metabolism , fevers and increased adrenocortical activity . It is decreased in the last stages of severe hepatic diseases and in acidosis .

Creatinine

It is derived from the creatin of the muscle and the amount excreted ( 1.0 to 1.8 g per day ) depends on the muscle mass

Analysis of Normal Urine

Physical Examination

Note the colour , appearance and odour . Test the acidity with blue or red litmus paper . Determine the specific gravity using urinometer . Apply the correction for temperature variation . Calculate the total solids present and perform the following tests with the sample .

Test for chlorides

Place 5 ml of albumin free test tube and add 3 drops of Con HNO3 to prevent precipitation of protein . Add 5 drops of AgCl indicates presence of chlorides .

Test for phosphates

To 3 ml of urine add 1ml of Con HNO3 and 5 ml of ammonium molebdate and warm . A canary yellow precipitate indicates the presence of phosphate.

Test for inorganic sulphates

To 3ml of urine add 1 ml of Con HCl followed by 10% BaCl2 solution .A white precipitate of BaSO4 indicates inorganic sulphate .Filter and set aside the filtrate for the next experiment.

Test for ethereal sulphate

Take the above filtrate and boil . A white precipitate is formed again indicating the presence of ethereal sulphate .

Test for ammonia

Boil urine with equal volume of 10% NaOH. Smell of ammonia gas indicates the presence of ammonium ion in the urine sample.

Analysis of Pathological Urine

In the case of abnormal urine , glucose , ketone bodies , proteins , blood , bile salts and bile pigments are tested in the clinical biochemical laboratory .

Test for Reducing Sugar. Benedicts Test

Reducing carbohydrates like glucose reduces CuSO4 in alkaline media to insoluble Cu2O . Colour of the solution depends upon the particle size of Cu2O formed .

Procedure

Take 5ml of Benedicts qualitative reagent . Boil for a minute and add 8 drops (0.5ml) of urine , boil for 2 minutes , cool and note the colour change .

Green 0.5 % sugar

Yellow 1.0%

Reddish yellow 1.5%

Brick red 2.0 % and above .

Test for Proteins (Albumins)

Heat and acetic acid test .Proteins are coagulated by boiling , CO2 driven out of the solution , pH of the urine is increased which may precipitate phosphates . Thus when a sample of urine is heated precipitate may be due to protein or phosphate addition of a few drops of acetic will dissolve phosphate , if present .

Procedure

Fill up 2/3 of a test tube with urine . Boil the upper portion of the test tube . Add a drop or two % acetic acid . A white cloud in the heated portion shows the presence of albumin .

Heller's Nitric Acid Test (Cold test )

This sensitive test is based on the fact that protein is converted into a meta protein which is insoluble in concentrated mineral acids .

Procedure

Take 3 ml of Con HNO3 in a test tube and pour 2 ml of urine along the sides . A white ring at the junction of the two layers shows the presence of proteins .

Sulposalicylic Acid Test

Place 1 ml urine in a test tube , add 3%bsulposalicylic acid and allow to stand for 10 min . Presence of cloudiness is due to albumin .

Benzidine Test for Blood in Urine

Prepare a saturated benzidine solution in glacial acetic acid ; mix well equal parts of benzidine solution and H2O2 . Take 2 ml of urine and add 2 ml of this mixture . A green or deep blue colour indicates presence of blood in urine . Any catalyst or enzyme which splits up H2O2 and liberates nascent oxygen will give this test . The pus cells ( WBCs in urine ) contain an enzyme called peroxidase , which can spilt H2O2 and liberate nascent oxygen . To test haemoglobin in presence of pus cells , the urine must be boiled to destroy the pus cells enzyme

Normally bile salts and bile pigments do not enter in the general circulation , and therefore , they are absent in normal urine . But if there is intrahepatic or extrahepatic obstruction to the flow of bile ,bile regurgitates into the general circulation and appears in urine as in obstructive jaundice . Bile pigments give urine a greenish yellow or brown colour .

a. Hays test for salts

When powder of sulphur is sprinkled on the urine containing bile salt, the sulphur powder sinks due to lowering of surface tension. A positive test indicates the liver damage or obstruction of bile duct.

b. Gmelin's test for bile pigments

When Con HNO3 is added to urine, there is a play of colour. Green indicates presence of bile pigments. Nitric acid oxidisses bilirubin to form a series of coloured compounds, biliverdin (green), billicyanin (blue), choletelin (Yellow), etc.

c. Fouchet's test for bile pigments

Add 5ml of 10% BaCl2 solution to about 10 ml of urine . Mix and filter . Pour a few drops of Faucets' reagent ( FeCl3 the oxidizing agent ) on the precipitate in the air dried filter paper . In the presence of bile pigments , bilirubin turns green and blue due to oxidation (UBG and SBG ) .

d. Ehrlichs aldehyde test for urobilinogen

Take 10 ml of urine in a test tube . Add 2.5 ml of BaCl2 solution and filter . Take 3 ml of filtrate add Ehrlich's aldehyde reagent (para dimethyl aminobenaldehyde) shake well and allow to stand for 3 minutes. Pink colour indicates a positive test.

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