BIOCHEMISTRY I

Lecture 5Lecture 5

The Kidney and Urinalysis Part III

Test for Renal Function

Urinalysis

3 parts examination (physical, chemical &

microscopic)

Creatinine and blood urea nitrogen (BUN) Creatinine and blood urea nitrogen (BUN)

Creatinine clearance test

Chemical Examination of Urine

Several biochemical tests can be performed

quickly and easily on urine using reagent strips

Chemical testing by reagent strip = chemical,

biochemical or analyte analysis of urinebiochemical or analyte analysis of urine

Reagent strips are used by dipping it in the

urine and observing chemical reactions on

each reagent pad within a specified time

Principle of urine Chemical Tests by

Reagent Strip

They are sometimes called dipstick

The parameters commonly tested are:

Glucose - Urobilinogen

Bilirubin - Nitrite

Ketones - Leukocyte esterase

Blood - Specific gravity

pH protein

It is best to prefer to manufacturers instructions since use of biochemical detection methods may differ

Glucose

Presence of detectable glucose in urine = glycosuria (indicates blood glucose level has exceeded the renal threshold for glucose)

Can occur in diabetes mellitus and gestational Can occur in diabetes mellitus and gestational diabetes

Reagent strip is specific for ONLY glucose

It contains enzymes glucose oxidase and peroxidase with a chromogen (a substance that produces a colour in chemical reaction)

Glucose

These enzymes react with glucose in the urine

- change colour of the reagent pad

Intensity of the colour formed - proportional

to the glucose concentrationto the glucose concentration

Normal urine is negative for glucose by this

method

Bilirubin

This is the primary bile pigment, formed from

the breakdown of haemoglobin

When present, it indicates:

Liver disease Liver disease

Bile duct obstruction

Hepatitis

Bilirubin

Test for bilirubin - based on coupling bilirubin

with a diazonium salt in the reagent pad to

form a purple - brown colour

Direct light causes decomposition of bilirubin Direct light causes decomposition of bilirubin

Thus, specimens should be protected from

light - prevent false - negative results

Normal urine contains no detectable bilirubin

by this method

Ketones

Ketones are formed when body burns fat

rather than sugar for energy

Ketones include:

Acetone Acetone

Acetoacetic acid

Beta-hydroxybutyric acid

Ketones

Ketonuria (presence of ketones in urine) may be caused by:

Uncontrolled diabetes

Starvation Starvation

Prolonged dieting or fasting

Ketone test - based on reaction of ketones with sodium nitroferricyanide in ketone reagent pad - cause formation of dark pink to maroon colour

Ketones

Ketones evaporate at room temperature - thus

must be tightly capped and refrigerated, if

cannot be tested promptly

Normal urine - negative for ketones Normal urine - negative for ketones

Blood

Presence of blood in urine = haematuria

Can be due to infection, trauma to the urinary tract, bleeding in the kidneys, glomerular damage or tumour

May be due to menstruation, trauma from catheter (a hollow flexible tube for insertion into a body cavity, duct, (a hollow flexible tube for insertion into a body cavity, duct, or vessel to allow the passage of fluids or distend a passageway. Its uses include the drainage of urine from the bladder through the urethra or insertion through a blood

vessel into the heart for diagnostic purposes) insertion

Blood

Blood reagent pads detects haemoglobin and

intact RBC

When either is present, a colour forms

because of the peroxidase - like action of because of the peroxidase - like action of

haemoglobin in the RBC reacting with

chromogen and peroxide in the reagent pad

Resulting colour range form orange to green

to dark blue

Blood

Intact RBC can cause a spotty appearance on

the reagent pad

Myoglobin - also cause a positive reaction on

the reagent stripthe reagent strip

Normal urine - negative

pH pH of urine changes with: Diet

Medications

Kidney disease

Metabolic diseases (e.g. diabetes mellitus)

Indicator dyes (e.g. methyl red and bromothymol Indicator dyes (e.g. methyl red and bromothymol blue) in the pH reagent pad forms colour from yellow - orange for acid urine to green - blue for alkaline urine

Normal urine = pH 4.5 - 8

Protein

Proteinuria - increased amount of protein in

urine

Proteinuria is important indicator of renal

disease but can be caused by vigorous disease but can be caused by vigorous

exercise or conditions such as urinary tract

infection (UTI)

Positive tests - caused by presence of albumin

Protein

Protein reagent pad - not a good indicator of

globulins

Protein sulfosalicylic acid turbidity test should

be used if proteins can alter colour of some be used if proteins can alter colour of some

acid - base indicator dyes without changing

the pH

The pad is kept at pH 3 with a buffering dye

(e.g. tetrabromophenol blue)

Protein

At the constant acid pH, development of any green

colour on the reagent pad is due to presence of

protein (albumin) and is usually reported using a

plus system (Neg, 1+, 2+, 3+ & 4+)plus system (Neg, 1+, 2+, 3+ & 4+)

Colour range form yellow for negative to yellow-

green or green for positive, depending on the

amount of protein present

Normal urine - negative or just a trace of protein

Urobilinogen

Is a bilirubin degradation product - formed by

action of intestinal bacteria

It can be increased in hepatic disease or

haemolytic diseasehaemolytic disease

Urobilinogen reagent pad contains chemicals -

react with urobilinogen to form a pink - red

colour, based on Ehrlich aldehyde reaction

Normal urine - 0.1 - 1.0 mg/dL (0.1-1.0 EU/dL)

Urobilinogen

Since urobilinogen - unstable in light and in

acidic urine, negative results - not considered

significant

Reagent strip can detect urobilinogen in Reagent strip can detect urobilinogen in

concentration as low as 0.1 mg/dL (0.1 EU,

Ehrlich unit)

Normal urine - 0.1 - 1.0 mg/dL (0.1 - 1.0 EU)

Nitrite

Gram-negative bacteria produce enzymes that convert urinary nitrate (a normal urine constituent) to nitrite

Nitrite reacts with chemicals on reagent pad to form a pink colourform a pink colour

Positive nitrite test is an indication of possible bacterial UTI

Since not all bacteria can convert nitrates, a negative result is possible in the presence of infection

Nitrite

Organisms which normally cause UTI and

cause a positive nitrite test are:

Escherichia coli

Klebsiella Klebsiella

Proteus

Pseudomonas

Normal urine - negative for nitrite by reagent

strip method

Leukocytes Esterase

Granular leukocytes (primarily neutrophils)

contain an enzyme called leukocyte esterase

Presence of this enzyme indicates presence of

leukocytes in urineleukocytes in urine

This may due to infection or inflammation in

the urinary tract

Leukocytes Esterase

Esterase enzyme reacts with esterase

substrates in leukocyte esterase reagent pad -

form a purple colour

Colour intensity is proportional to number of Colour intensity is proportional to number of

leukocytes present

Normal urine - negative result for this test

Specific Gravity

Specific gravity reflects kidneys ability to

concentrate urine

The pad contains indicator which change from

blue-green to yellow-green with relevance to blue-green to yellow-green with relevance to

urine ion concentration

Normal specific gravity for urine is 1.005 to

1.030 by reagent strip method

Reference Values by Reagent Strip

Normal urine:

Negative for glucose, ketone, bilirubin, bacteria

(nitrite), leukocyte esterase and blood

May be negative or contain trace amount of May be negative or contain trace amount of

protein

Usually have pH of 4.5 8.0 and specific gravity of

1.005 - 1.030

Positive or abnormal results should be

confirmed according to lab policy

Positive leukocyte esterase or nitrite tests

should be confirmed by microscopic should be confirmed by microscopic

examination of urine sediment

Single Analyte and Special Reagent Strips

Reagent strips that test for only 1 or 2

constituents (e.g. glucose and ketones) -

useful for monitoring purposes

Examples: Examples:

Diastix and Clinistix - used at home to monitor

urine glucose

Ketostix - only test for ketones

Keto-Diastix - for ketones and glucose

Single Analyte and Special Reagent Strips

Multistix PRO - to detect or monitor kidney

disease and diabetes mellitus, also measure

creatinine and protein

Clinitek Microalbumin - detect microalbumin

(protein in the urine in amounts too small to be

detected by routine strips)

Microalbuminuria = condition in which very small

amounts of albumin are present in the urine

This indicates early renal disease and can occur as a

complication of diabetes

Performing Chemical Tests by Reagent Strips

Directions for use and colour comparison

chart are included with each vial

Instructions must be followed for accurate Instructions must be followed for accurate

results

Safety Precautions

All specimens must be considered potential

biological hazards

Gloves and face shields are best to be worn

when handling these specimenswhen handling these specimens

Avoid splashes, spills and creation of aerosols

Quality Assessment

Specimen Collection and Storage

Specimens must be collected and labeled

correctly and tested within required time

limitslimits

Midstream specimen is preferred

Testing should be done within 1 hour of

collection and can be refrigerated up to 4

hours otherwise

Quality Assessment

Specimens should be allowed to reach room

temperature before testing and should be

tightly covered in the dark because urine

components are volatile, labile or light components are volatile, labile or light

sensitive

Quality Assessment1. Reagent Strips

Manufacturers directions for proper storage, use, timing of reactions must be followed to ensure validity of results

Strips must be stored protected from light, heat and moisture

They must not be used after expiration date and used only They must not be used after expiration date and used only once

Specimens must be mixed well ( make sure all solid components will be exposed to the pads

Test are based on colour comparison - technicians must pass colour blindness test

Quality Assessment2. Urine Control

Reliability of urine reagent strips validated by urine chemistry controls ensure the integrity of strips

Urine controls should be run at least once each shift (or day)(or day)

Commercial normal (-ve), low abnormal and high abnormal controls should be used

Patients results should not be reported if the strip does not show valid results based on the manufacturers stated ranges

Manual Method Test is performed by quickly

dipping a reagent strip into recently collected, well mixed urine

Timing of reactions must start when the strip is withdrawn from when the strip is withdrawn from the urine

Colour changes on pads must be visually compared to the colour chart provided by the manufacturer

Manual Method

Reagent Strip Readers

Are used to interpret the results of urine reagent strip tests

The readers contain reflectance photometers -detect colour formed on the reagent pads

Results may be read out from the display screen Results may be read out from the display screen or print out

Reduces errors caused by

incorrect timing of reaction

or interpretation of colours

Performing Copper Reduction Test - Clinitest

Copper reduction test - detects reducing

sugars (glucose, fructose, lactose, galactose)

Substances which may give false-positive

reactions with this test are:reactions with this test are:

Penicillin

Salicylates

Ascorbic acid

Cephalosporins

Performing Copper Reduction Test - Clinitest

Previously, Clinitest was used to estimate

glucose in urine of individuals with diabetes

But with glucose-specific reagent strips today,

this method is no longer used for glucosethis method is no longer used for glucose

Copper reduction test - now used as a simple

way to screen urine for reducing sugars other

than glucose (e.g. screen newborns for

galactosuria = excretion of galactose in urine)

Performing Clinitest

5 drops of urine and 10 drops of water are added into heat - resistant test tube

Clinitest tablet is placed in the diluted urine -colour is observed while it effervesces

If a reducing sugar is present, the colour changes If a reducing sugar is present, the colour changes from blue to green and then to orange -depending on amount of reducing substance present

When the reaction is complete, colour of the liquid is compared to the colour chart on the vial

Performing Confirmatory Tests

Methods other than reagent strips - needed to confirm a positive (or negative) result

Confirmatory tests - more time consuming, require more reagents and equipments than require more reagents and equipments than reagent strip method

Commonly performed tests:

Proteins

Ketones

Bilirubin

Protein

Most simple confirmatory tests for urine

involve treating a portion of the urine with an

acid - cause protein to precipitate and become

visiblevisible

Amount of precipitate proportional to

concentration of proteins present

Protein

Precipitate is graded as:

Negative

Trace (slightly cloudy)

1+ (turbid) 1+ (turbid)

2+ (turbid with granulation)

3+ (granulation and flocculation - lumps / fluff)

4+ (clumps)

Dilute acetic or sulfosalicylic acids can be used to precipitate protein

Ketones

Acetest - test for ketones and is available in

tablet form

If ketones are present, a drop of urine will

produce a purple colourproduce a purple colour

Serum or plasma - used to test for ketones

using Acetest tablet

Ketostix and Keto-Diastix - also used to

confirm presence of ketones in urine

Acetest

Bilirubin

Ictotest - specific test for bilirubin and 4x more

sensitive as reagent strip method

A tablet and absorbent mat /paper is used - a

few drops of urine is placed on the mat, tablet few drops of urine is placed on the mat, tablet

is positioned on the moist area and water is

dropped on the tablet

Purple colour will develop within 60 seconds,

if bilirubin is present

Ictotest