DIAGNOSTIC IN CLINICAL

CHEMISTRY I
MKEB 2404

EXAMINATION OF BODY FLUIDS

Type of body fluids:
 Urine
 Seminal fluid
 Amniotic fluid
 Cerebrospinal fluid
 Synovial fluids
 Pleural, pericardial and peritoneal fluid
 Fecal analysis

First morning urine collection:

 Voids before going to bed
 Formed elements are more stable
 Unsuitable for cytology studies
 Collection of midstream urine for avoid contamination (let the first urine
throw first and only take the middle urine)

Urine container:
 Wide mouth (4 - 5 cm)
 Sufficient volume (50 ml preferred)
 Glass or plastic with no additives
 Leak-proof
 Sterile, if specimen is stored for a period of time before testing

The importance of urinalysis: as an indicator of health or disease, especially with

metabolic and renal disorders

Potential changes in unpreserved urine:

Physical changes
Bilirubin - biliverdin
Color Hemoglobin - methemoglobin
Urobilinogen - urobilin
Clarity Decreased due to bacterial proliferation,solute precipitation
Odour Increased due to bacterial proliferation and decomposition
Chemical changes

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 pH Increased or decreased
Glucose decreased
Ketones decreased
Bilirubin decreased
Urobilinogen decreased
Nitrite Increased or decreased
Microscopic changes
 Decreased due to disintegration especially in alkaline
urine
RBC, WBC, casts  RBC decreased after 6 hours
 WBC decreased 50% within 3 hours
 Hyaline and granular casts decreased after 2 hours
Bacteria Increased due to bacterial proliferation
 Precipitation of uric acid, calcium phosphate and
calcium oxalate
 Yeast cells develop pseudo-mycelia
Others  Spermatozoa become immobile
 Trichomonas become immobile, maybe counted as
WBC
 Contamination by air borne particles

Urine examination:
 Most preservatives prevent bacterial growth and loss of glucose (eg.
Stabilur, formalin)
 No preservatives can prevent destruction of bilirubin, urobilinogen or
occult blood.
 Use of preservatives may increase SG, minor effects on pH and may
inhibit leukocyte esterase reaction.
 No single urine preservative is available

Urine FEME:
Physical examination
Color  Urochrome, urobilin, uroerythrin
 Normal color range from straw, pale yellow, to amber.
Abnormal color:
 Red - RBCs
 Beer-brown - bilirubin
 Orange, blue, green - drug, dye or food
 Colorless - dilute urine - fluid ingestion: polyuria
 Light yellow and yellow - normal Urine
 Amber - concentrated urine - dehydration, fever

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  Amber – urobilin - no yellow foam
 Dark amber – bilirubin - yellow foam
 Dark amber – biliverdin - imparts green blue
 Orange – bilirubin - yellow foam if sufficient bilirubin
 Orange – urobilin - no yellow foam
 Orange – medication
 Red - hemoglobin, red blood cell
 Red – myoglobin - muscle injury
 Red - porphyrins
 Red – beets - genetic
 Red - fuscin, analine dye - food, candy
 Pink – hemoglobin, porphyrins
 Brown – hemoglobin, myoglobin, methemoglobin
(muscle), homogentisic acid (acid pH), melanin
 Black – melanin (upon standing), homogentisic acid
(upon standing : alkaline urine)
 Green blue – indican (infections of small intestines),
chlorophyl (breath deodorizers), pseudomonas
infection, dyes and medication
Color changes due to oxidation:
 RBC oxidizers (brown) → methemoglobin (black)
 Urobilinogen (colorless) → urobilin (orange – brown)
 Porphobilinogen (colorless) → oxidizers to
porphobilin (red / purple)
 Bilirubin (amber) → oxdizers to biliverdin (greenish)
Urine color changes with commonly used drugs:
 Alcohol, ethyl – pale, diuresis
 Anthraquinone laxatives – reddish – alkalinr, yellow
brown – acid
 Chlorzoxazone (muscle relaxant) – red
 Deferoxamine mesylate (desferal) – red
 Furazolidone (an antibacterial, anti protozoal
pitrofuran) – brown
 Indigo carmine dye (renal function, cytoscopy) – blue
 Iron sorbital (jectofer) – brown on standing
 Leyodona (parkinsonism) – red then brown, alkaline
 Alcohol – pale
 Desferal and paraflex (muscle relaxant) – red
 L – Dopa (parkinsonism) – red then brown
 Flagyl – reddish brown
 Nitrofurantoin – brown – yellow

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  Riboflavin – bright - yellow
Clear, slightly cloudy, cloudy, turbid
Turbidity or cloudy urine:
 Amorphous salts – non pathologic
 Bacteria, blood cells - pathologic
Causes of turbidity (pathologic):
 RBC, WBC
 Bacteria, yeast, trichomonas
 Renal epithelial cells
Clarity and
 Fat (lipids, chy;e)
Odour
 Abnormal crystals, calculi and pus
Causes of turbidity non pathologic:
 Normal crystals like urates and phosphates
 Radiographic media
 Mucus, mucin, squamous epithelial cells
 Sperm, posthatic fluid
 Salves, lotions, cream
 Powders, talc
Chemical examination
 Volume - average of 1.0 to 1.5L of urine excreted per
day
 Amount excreted is an indicator for diuretic disorder
Urinalysis  Polyuria: More than 2000ml urine/day
 Oliguria: Less than 500ml urine/day
 Anuria : Less than 200ml urine/day
 Dysuria: No urinary excretion
 Example is Bayer – ames multistix
 Manual – subjective
Dipstick
 Machine – standardized reflectance photometer that
methods
measures scattered or reflected light, have multiple
channels and compensator pad
 Store in original container
Dipstick  Do not expose to light, heat and moisture
methods – care  If there is any colour change, discard
and storage  Do not use pass expiration date.
 Store at manufacturer recommended temperatures
Dipstick – testing  Well-mixed uncentrifuged urine sample
procedure  Dip strip into urine briefly
(manual)  Remove excess urine
 Read colour development according to manufacturer’s
instruction

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  Read in a well lit area
 Aware of false positive and false negative results
Reaction:
 Pseudoperoxidase action of Hgb myoglobin catalyzes
the oxidation of chromogens to produce a color change
False negatives:
 Formalin, excess nitrites (>2.2 mmol/l), elevated SG,
ph <5.1, captopril, ascorbic acid
False positive:
 Oxidizing detergents, microbial peroxidase (UTI),
dehydration, exercise, hemoglobinuria,
myoglobinuria, menstrual contaminants, proteinuria
(>5 g/l)
Hematuria:
 Presence of an abnormal number of blood cells in
urine as microhematuria or gross hematuria (0.5ml or
2500 RBC/µl)
 Occurs with disease or trauma anywhere in the
Blood
kidneys or urinary tract
 Can be seen in healthy persons undertaking excessive
exercise (marathon runners) in whom bleeding
emanates from the bladder mucosa. Repeat urinalysis
after 48 – 72 hours should be negative
 Causes: cancer, trauma, stones, infections,
obstructions, viral infections, inflammation of kidneys,
benign prostate enlargement, and warfarin therapy
 Calculi – ca oxalate (60%), uric acid (25%) phosphate
(20%)
 Tumors – painless hematuria
 Glomerulonephritis – hematuria with proteinuria
 Urinary tract infection
Separate sales:
 Green dots (intact RBC)
 Homogenous green color scale (for lysed RBC)
Bilirubin Reaction:
 Bilirubin in the urine couples with a diazonium salt in
an acid medium
False negative:
 Samples exposed to light, excess levels of ascorbic
acid.and nitrite, selenium, chlorpromazine
False positives:
 Highly colored metabolites of drugs eg pyridium

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 _____________________________________________________
 Breakdown product of hemoglobin formed in the in
the RES, liver, and bone marrow
 carried in the blood by protein
 Normal adult urine contains 1 mg/dL and this is not
detected by usual tests.
Reaction:
 Double sequential enzyme reaction of glucose oxidase
and peroxidase-reacts with a chromogen to produce
the final color.
False negative:
 Elevated specific gravity, uric acid, ascorbic acid
False positives:
Glucose  presence of oxidizing agents, ketones, levodopa
_____________________________________________________
 May appear in the urine and is influenced by:
o Blood glucose levels
o Glomerular blood flow
o Tubular reabsorption rate
 Often regarded as a hallmark of disease and requires a
patient to receive a workup for diabetes mellitus.
Reaction (Legal or rpthera’s test):
 Reaction with nitroprusside or sodium
nitroferricyanide and glycine to produce a color
change.
 β-hydroxybutyerate 78%, acetoacetate 20%,acetone 2%
False negative:
 Delay in examination
False positives:
Ketones
 Highly pigmented urines; some drug metabolites,
acidic urine, elevated SG
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 Products of incomplete fat metabolism
 Presence is indicative of acidosis
 Low carbohydrate diet for weight reduction will
produce ketonuria
 Exposure to cold and severe exercise
Leukocytes Reaction :
 Leukocyte esterase, present in granulocytes, catalyzes
the reaction of the chromogens to produce a color
change.
False negative:

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  Cephalexin and gentamicin concentrations; elevated
SG, glucose, ketone and protein concentrations,
ascobic acid
False positives:
 Vaginal contaminants, drugs or foods that color the
urine red
Reaction :
 Nitrates in the urine are converted to nitrites by the
action of gram-negative bacteria. These nitrites then
react to form a diazonium salt which in turn reacts
with a chromogen to produce the final color.
Nitrites False negative:
 Elevated SG, urobilinogen, pH <6.0, excess ascorbic
acid
False positives:
 Presence of red dyes or other chromogens,
contamination
pH Reaction:
 Double indicator system detects the amount of
hydrogen ions in the urine to produce a color change.
Interferences:
 If excess urine is left on the reagent strip, a
phenomenon known as “runover” may occur. The
urine from one reagent area carries reagent onto the
pH test area and changes the result erroneously.
 Reflection of the ability of the kidney to maintain
normal hydrogen ion concentration in plasma and
extracellular fluid
 Normal adult: 4.6 - 8.0 pH
o Hypertonic urine < 6.0 - crenated RBC
o Hypotonic urine > 7.5 - Lysis of cells
 Acid urine: diet high in meat protein
 Alkaline urine: diet high in citrate or vegetables
 RTA type I (renal tubular acidosis)
o Serum is acidic, urine is alkaline
 RTA type II
o Urine initailly alkaline but becomes more acidic
due to decrease in bicarbonate load
 Useful in diagnosis and management of UTI and
calculi
o Alkaline urine in UTI suggests presence of urea-
splitting organisms

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 o Magnesium-ammonium phosphate crystals can
form staghorn calculi
o Uric acid calculi associated with acidic urine
Reaction :
 Based on “protein error of indicators” - because
protein carries a charge at physiologic pH, their
presence will elicit a pH change
False negative:
 Acidic or diluted urine, primary protein is not albumin
False positives:
 Alkaline or concentrated urine, quaternary ammonia
compounds
Protein
 High levels in urine indicates renal disease:
o Glomerular disease
o Tubular disease
 Functional proteinuria
o After strenuous exercise
Other methods of detection:
 Heat
 Acid (SSA- sulfosalicylic acid precipitation test)
Sensitivity: 5-10 mg/dL of protein
Reaction : ionic specific gravity
 Based on the change of an indicator color in the
presence of high concentrations of various ions.
False negative:
 Highly alkaline urine
False positives:
 Proteinuria, Dextran solutions,IV radiopaque dyes,

Specific gravity  Random SG - 1.015 - 1.025

 SG< 1.010 indicates relative hydration
 SG > 1.020 indicates relative dehydration
 SG - 1.000 should be checked
 SG - 1.040 physiologically impossible
 Other methods: urinometer and refractometer
 Correlates with urine osmolality
 Insight to patient’s hydration status
 Reflects concentration ability of the kidneys
Urobilinogen Reaction : ionic specific gravity
 Urobilinogen reacts with a chromogen to form an azo
dye which appears as various shades of pink or

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 purple.
False negative:
 Excess nitrites; presence of formalin
False positives:
 Presence phenazopyridine; very warm urine, elevated
nitrite levels

 Elevated urobilinogen found in hemolysis and

hepatocellular diseases
 Decrese robilinogen levels can be due to antibiotic use
and bile duct obstruction
 Leucocyte
 RBC
Test strip sieve
 Protein
technique
 Nitrite
 pH > 7.0
Microscopic examination
 Most common laboratory procedure utilized for the
detection of renal and/or urinary tract disease
Sediment  Numerous morphologic entities
examination o Blood cells, epithelial cells, organisms
 Correlate with the biochemical results
o Dipsticks
o Clinical condition of the patient
 Cells
o Blood cells; RBCs and WBCs
o Epithelial cells; renal, transitional, squamous
 Casts
Microscopic
o Hyaline
components in
o Waxy
urine sediment
o Inclusion casts; Granular, Fatty
o Cellular; RBC, WBC, and Epithelial casts
 Bacteria, Fungi, and Parasites
 Crystals
 Bright field Microscopy of unstained urine and w/
Supravital staining
Methods for  Phase Contrast Microscopy
examining urine  Polarized Microscopy
sediment  Interference Contrast Microscopy
 Cytodiagnostic Urinalysis
 Quantitative and Differential Counts

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  Appear as pale discs
 Can be confused with yeast cells
Red blood cells
 Yeast cells do not stain and are not lysed by the
addition of acetic acid
 Increased numbers in conjunction with RBC cast
Dysmorphic bleeding assumed to be renal in origin
RBCs  Absence of casts and protein - bleeding assumed to be
non-renal
 Increased numbers are seen:
o Renal diseases
Leukocytes o Urinary tract infection
 When accompanied by casts:
o Renal in origin
 Line the distal 1/3 of the urethra
Squamous
 Large numbers in women maybe a source of
epithelial cells
contamination
 Line the urinary tract from the renal pelvis to the
Transitional
proximal 2/3 of the urethra
epithelial cells
 few are present in normal urine
 Small numbers maybe seen in normal urine
o Sloughing of aging cells
Renal tubular
 Increased numbers are seen:
epithelial cells
o Acute tubular necrosis
o Certain drug or heavy metal toxicity
 Formed when an increased numbers of proteins enter
the tubules.
 Formation increases with:
o Lower pH
o Increased ionic concentration
 Tamm-Horsfall (TH) protein forms the matrix of all
casts
Casts
o Glycoprotein secreted by cells in the ascending
loop of Henle
 If cast contain 3 or more cells e.g. RBC, WBC, then it is
RBC cast, WBC cast
 If it contains 1/3 or more granules - granular cast
 If a cast is about 60 µm or more - broad cast, RBC 7-8
µm, WBC 8-22 µm
Hyaline cast  Translucent with brightfield microscopy
 Increased numbers:
o Pyelonephritis, chronic renal disease

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 o Transiently with exercise
o May be a normal finding
 Associated with tubular inflammation and
Waxy cast degeneration
 Observed frequently with chronic renal failure
 Appear with glomerular and tubular diseases
 Accompany:
Granular casts o Pyelonephritis
o Viral infections
o Chronic lead poisoning
 Commonly seen when there is heavy proteinuria
Fatty casts  A feature of nephrotic syndrome
 Hypothyroidism
 Diagnostic of glomerular disease
Red blood cells
 Glomerular damage allows RBCs to escape into the
casts
tubules
 WBCs enter the tubular lumen through and between
White blood cells tubular epithelial cells
casts  Associated with pyelonephritis and tubulointerstitial
disease
 To differentiate from leukocyte cast, supravital
Epithelial cells staining and phase -contrast microscopy are helpful.
casts  Associated with: tubular necrosis, viral disease (CMV),
heavy metal ingestion
Other component that also found is bacteria, fungi, and parasites and also clue
cells
 Limited clinical significance
 Phosphates, urates, and oxalates are common and
occur in normal urine
 Alkalization and refrigeration promotes crystals
formation
 Few crystals are important:
Crystals o Cystine
o Tyrosine
o Leucine
o Bilirubin - hepatic and biliary tract diseases
o Cholesterol - nephrotic syndrome
 Cysteine, leucine and tyrosine is due to inherited
metabolic disorders
Crystals found in  Amorphous urates / phosphates
normal urine  Calcium oxalates

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  Uric acid
 Triple phosphates
 Cysteine
Crystals found in  Bilirubin
abnormal urine  Tyrosine
 Cholesterol
Procedure for Sample collection → centrifugation → decantation → slide
urine microscopy preparation → microscopy → writing report
Centrifuge or not to centrifuge:
“After a 5 min centrifugation of the urine at 3500rpm, only 48%
Quantitative ME
of RBC and 40% of WBC found to be present could still be detected
(microscopic
under the microscope”
examination)
To report in µl or LPF or HPF
To stain or not to stain
Neubauer See picture below:
counting
chamber
 Analysis volume of Neubauer is half of Fuch
Rosenthal
o Concentration of urine formed elements is very
Fuch Rosenthal
low compared with those of hematology.
vs Neubauer for
o For urinalysis, the more volume, the better
quantitatve urine
results
microscopy:
 Some urine formed elements are large (ie casts), these
Advantages of
might clog the chamber
Fuch Rosenthal
o The deeper the depth, the better
over Neubauser:
 Cells /uL = total cells counted / [mm2 counted (how
many mm squares were conted) x 0.1 mm (neubauer
chamber) or 0.2mm (fuch rosenthal chamber)]
Depends on:
 Real View ( Diameter )
 Magnification# ( x10 or x40 )
HPF LPF or µl
 Original Urine Volume before Centrifugation
conversion
 Sediment Volume after Centrifugation
 Loaded Sediment Volume on the Slide
 Area of Cover Slip
Procedure for Low power microscopy:
microscopic  Ensure uniform distribution of urine sediment
examination  If uneven distribution, make a new preparation
 Reduce light intensity
 Scan whole area. Note: sediments tend to gather along
sides of cover-slip

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 High power microscopy:
 Examine 20 - 30 fields (optimal) but not less than 10
fields
Microscopic with staining sediment to stain 4:1
Types of stain:
1. Sternheimer-Malbin Stain (SM Stain)
2. Sterheimer Stain (S Stain)
3. 0.5% Toluidine Blue
4. Sudan III and Oil Red O
Blood cells:
 Less than 1cell/HPF
 1 - 4 cells/HPF
 5 - 9 cells/HPF
 10 - 19 cells/HPF
 20 - 29 cells/HPF
 30 - 49 cells/HPF
 50 - 99 cells/HPF
 Numerous -100 cells and more
Casts:
- :0
Procedure for + : 1 cast/100LPF or 1 cast/WF
microscopic ++ : 1 cast/LPF or 100 casts/WF
examination +++ : 10 casts/LPF or 1,000 casts/WF
reporting format: ++++ : 100 casts/LPF or 10,000 casts/WF
or 6 casts/HPF
Bacteria and yeast:
- :0
+/- : scatter in several fields
+
++
3
: seen in each foeld
: many or scatter in cluster
+++ : numerous
Crystals and amorphous materials:
- :0
1 +
++
W : 1 ~ 4/HPF
: 5 ~ 9/HPF
W
+++: 10 ~ /HPF

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W W
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