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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