Exa minati on of

Body F luid s
Woon Sung Thong, FMIMLS
Past President, Malaysian Institute of Medical Laboratory Sciences
Laboratory Manager, Biolife Lab Sdn Bhd
 Body Fluids

• Urine
• Seminal Fluid
• Amniotic Fluid
• Cerebrospinal Fluid
• Synovial Fluid
• Pleural, Pericardial and Peritoneal Fluid
• Fecal analysis
 Urine Collection

First morning
*voids before going to bed
*formed elements are more stable
*unsuitable for cytology studies
Random
• ? Second voiding
 Collect ion
Techniques
Routine Midstream

Midstream “Clean Catch”

*for eliminating contamination
*for culture
 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
 Why bot her w ith
ur inaly sis ?

• At pre-employment medical
examinations
• Routinely at physician office and
medical clinics
• Annual medical examination

An indicator of health or disease,

especially with metabolic and renal
disorders.
What are the potenti al changes in
unpreserved u rine?
 Potentia l C hanges
in Unpre serv ed
Ur in e
Physical changes
Colour- bilirubin - biliverdin
hemoglobin - methemoglobin
urobilinogen - urobilin
Clarity - Decreased due to bacterial
proliferation,solute precipitation
Odour - Increased due to bacterial
proliferation and decomposition
 Potentia l C hanges
in Unpre serv ed
Urine
Chemical Changes
pH - increased or decreased
Glucose - decreased
Ketones - decreased
Bilirubin - decreased
Urobilinogen - decreased
Nitrite - increased or decreased
 Potentia l C hanges in
Unpres erv ed Urine

Microscopic Changes
RBC, WBC, Casts
*decreased due to disintegration
especially in alkaline urine
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
 Potentia l C hanges
in Unpre serv ed
Urine
Microscopic Changes
• Precipitation of uric acid, calcium
phosphate and calcium oxalate
• Yeast cells develop pseudo-mycelia
• Spermatozoa become immobile
• Trichomonas become immobile,
maybe counted as WBC
• Contamination by air borne particles
 Urine Examinatio n

Preservatives
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.
 Urine Examinatio n

No single urine preservative is available

NCCLS recommends that it be analyzed

within 2 hours. Refrigeration can induce
precipitation of amorphous urates and
phosphate crystals that can interfere
substantially with microscopic
examination
What is FE ME?
 Urine FEME

• Physical Examination

• Chemical Examination

• Microscopic Examination
 Urine - Phys ical
Examination
• Colour - urochrome, urobilin, uroerythrin
• Clarity
Clear
Slightly cloudy
Cloudy
Turbid
• Odour
 Physical Tests

• Color
Normal color range
from straw, pale
yellow, to amber.
Abnormal color:
red - RBCs
beer-brown - bilirubin
orange, blue, green
- drug, dye or food
 CO LO UR
ITEMS
Colour Constituent Comments

Colorless, Dilute urine Fluid ingestion: polyuria

Light Yellow
Yellow Normal Urine
Amber Concentrated urine Dehydration, fever
Urobilin No yellow foam
Dark amber Bilirubin Yellow foam
Biliverdin Imparts green blue
Orange Bilirubin Yellow foam if sufficient bilirubin
Urobilin No yellow foam
Medications
Red Hemoglobin, red blood cell
Myoglobin Muscle injury
Porphyrins
Beets Genetic
 COLOU R
IT EMS
Colour Constituent Comments
Pink Hemoglobin
Porphyrins
Brown Hemoglobin
Myoglobin
Methemoglobin Muscle
Homogentisic acid Acid pH
Melanin
Black Melanin Upon standing: rare
Homogentisic acid Upon standing: alkaline urine
Green blue Indican Infections of small intestine
Chlorophyll Breath deodorizers
Pseudomonas infectiion
Dyes and medications
 Colour change due to oxidation
RBC oxidizes methemoglobin
brown black
urobilinogen oxidizes urobilin
colourless orange-brown

porphobilinogen oxidizes porphobilin

colourless red/purple

bilirubin oxidizes biliverdin

amber greenish
 Ur in e colo ur changes with
commonly u sed d rugs
Drug Colour
Alcohol, ethyl pale, diuresis
Anthraquinone laxatives reddish-alkaline, yellow brown-acid
Chlorzoxazone Red
(muscle relaxant)
Deferoxamine mesylate Red
(Desferal)
Furazolidone Brown
(an antibacterial, anti protozoal nitrofuran)
Indigo carmine dye Blue
(renal function, cytoscopy)
Iron sorbital (Jectofer) Brown on standing
Levodopa (parkinsonism) Red then brown, alkaline
 Urine color changes with
commonly used drugs
• Alcohol • Pale
• Desferal • Red
• Paraflex (muscle relaxant) • Red
• L-dopa (for parkinsonism) • Red then brown
• Flagyl • Reddish brown
• Nitrofurantoin • Brown-yellow
• Riboflavin • Bright yellow
 Physical Tests

• Turbidity
Normal is essentially
clear.
Cloudy urine:
amorphous salts
- non pathologic
bacteria, blood cells
- pathologic
 Causes of Tu rbidity

Pathologic Non pathologic

RBC Normal crystals
WBC (eg. Urates, phosphates)
Bacteria Radiographic media
Yeast, Trichomonas Mucus, Mucin
Renal Epithelial Cells Squamous epithelial cells
Fat (lipids, Chyle) Sperm/prostatic fluid
Abnormal crystals Salves, lotions, cream
Calculi Powders, talc
Pus
Chemical
Examination
Ur in alysis -
Analysis technique
Urinalysis
Physical examinations
Volume - average of 1.0 to 1.5L of
urine excreted per day
Amount excreted is an indicator for
diuretic disorder
Polyuria: More than 2000ml
urine/day
Oliguria: Less than 500ml urine/day
Anuria : Less than 200ml urine/day
Dysuria: No urinary excretion
 Chemical
Examination
Dipstick method
(manual and machine)
Bayer- Ames Multistix
Roche - BM Combur 10
SD UroColor 11
Teco Diagnostics - URS - 10
Yeongdong - Uriscan - Gen 11
 Chemical
Examination
Dipstick method
Manual : Subjective
Machine : Sandardized
Reflectance photometer
measures scattered or reflected light
multiple channels (LED)
compensator pad
Instrumentation
2.5 Concentration Table - (Program Chip Card I)
The Miditron(R) Junior prints the test results in the following concentration ranges:
2.5 Concentration Table - (Program Chip Card I)
The Miditron(R) Junior prints the test results in the following concentration ranges:
Chemical examination
Dips ticks
 Dipstick - Care and
Storage
• Store in original container
• Do not expose to light, heat and moisture
• If there is any colour change, discard
• Do not use pass expiration date.
• Store at manufacturer recommended
temperatures
 Dipstick - Testing
Procedure (Manual)
• Well-mixed uncentrifuged urine sample
• Dip strip into urine briefly
• Remove excess urine
• Read colour development according to
manufacturer’s instruction
• Read in a well lit area
 Dipstick – Testing
Shortfall

• Aware of false-positive and false-

negative!
 Blood

• 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 positives:
– oxidizing detergents, microbial peroxidase (UTI),
dehydration, exercise, hemoglobinuria, myoglobinuria,
menstrual contaminants, proteinuria (>5 g/l)
 Blood

• 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
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
 Blood

• Separate scale is given:

- green dots (intact RBC)
- homogenous green color scale
(for lysed RBC)
 Blood

• Even one episode of hematuria must

be investigated

•Cancer •Inflammation of
•Trauma kidneys
•Stones •Benign prostrate
•Infections enlargement
•Obstructions •Warfarin therapy
•Viral infections
 Blood

• Calculi - Ca oxalate = 60%

Uric acid = 25%
Phosphate = 20%
• Tumors - painless hematuria
• Glomerulonephritis - hematuria with
proteinuria
• UTI
 Bilirubin

• Reaction:
– Bilirubin in the urine couples with a diazonium salt in an acid
medium
• False negatives:
– samples exposed to light, excess levels of ascorbic acid.and
nitrite, selenium, chlorpromazine
• False positives:
– highly colored metabolites of drugs eg pyridium
 Bilirubin

• 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.
 Glucose

• Reaction:
– double sequential enzyme reaction of glucose oxidase and
peroxidase-reacts with a chromogen to produce the final
color.
• False negatives:
– elevated specific gravity, uric acid, ascorbic acid
• False positives:
– presence of oxidizing agents, ketones, levodopa
 Glucose

• May appear in the urine and is influenced

by:
– blood glucose levels
– glomerular blood flow
– tubular reabsorption rate
• often regarded as a hallmark of disease
and requires a patient to receive a
workup for diabetes mellitus.
 Ketones

• Reaction: (Legal or Rothera’s test)

– Reaction with nitroprusside or sodium nitroferricyanide
and glycine to produce a color change.
β-hydroxybutyerate 78%, acetoacetate 20%,acetone 2%
• False negatives: - delay in examination
False positives:
– highly pigmented urines; some drug metabolites, acidic
urine, elevated SG
 Ketones

• 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 negatives:
– 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
 Nitrites

• 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.
• False negatives:
– 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.
 pH

• 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
- Hypertonic urine < 6.0 - crenated RBC
- Hypotonic urine > 7.5 - Lysis of cells
• Acid urine: diet high in meat protein
• Alkaline urine: diet high in citrate or vegetables
 pH
• RTA type I (renal tubular acidosis)
- serum is acidic, urine is alkaline
• RTA type II
- urine initailly alkaline but becomes more acidic due
to decrease in bicarbonate load
• Useful in diagnosis and management of UTI and
calculi
- alkaline urine in UTI suggests presence of urea-
splitting organisms
- magnesium-ammonium phosphate crystals can form
staghorn calculi
- uric acid calculi associated with acidic urine
 Protein

• Reaction:
– based on “protein error of indicators” - because protein
carries a charge at physiologic pH, their presence will elicit a
pH change
• False negatives:
- 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:
– Glomerular disease
– Tubular disease
• Functional proteinuria
– after strenuous exercise
 Protein

Other methods of detection:

• Heat
• Acid (SSA- sulfosalicylic acid
precipitation test)
sensitivity: 5-10 mg/dL of protein
 Specific Gravity

• Reaction: ionic SG
– based on the change of an indicator color in the
presence of high concentrations of various ions.
• False negatives:
– 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. Refractometer
 Specific Gravity

- Correlates with urine osmolality

- insight to patient’s hydration status

- reflects concentration ability of the kidneys

 Urobilinogen

• Reagent:
– urobilinogen reacts with a chromogen to form an azo
dye which appears as various shades of pink or purple.
• False negatives:
– excess nitrites; presence of formalin
• False positives:
– presence phenazopyridine; very warm urine, elevated
nitrite levels
 Urobilinogen

• Elevated urobilinogen found in hemolysis

and hepatocellular diseases

• Decrese robilinogen levels can be due to

antibiotic use and bile duct obctruction
 Ascorbic Acid
Interference with dipstick

Tests affected Ascorbic Acid Conc

Needed
Blood 9 mg/dl
Glucose 50 mg/dl
Bilirubin 25 mg/dl
Nitrite 25 mg/dl

Bridgen (1991): 22.8% positive (4379)

Mean: 37.2 mg/dl (7.1 - 333.5 mg/dl)
 “Test Strip Sieve Technique”

• Leucocyte
• RBC
• Protein
• Nitrite
• pH > 7.0
Micr oscopic
Examination
 Sediment examination

• Most common laboratory procedure

utilized for the detection of renal and/or
urinary tract disease
• numerous morphologic entities
– blood cells, epithelial cells, organisms
• correlate with the biochemical results
– dipsticks
– clinical condition of the patient
 Microscopic Components
in Urine Sediment

• Cells
– blood cells; RBCs and WBCs
– epithelial cells; renal, transitional, squamous
• Casts
– Hyaline
– Waxy
– Inclusion casts; Granular, Fatty
– Cellular; RBC, WBC, and Epithelial casts
• Bacteria, Fungi, and Parasites
• Crystals
 Methods for Examining
Urine sediment

• Bright field Microscopy of unstained

urine and w/ Supravital staining
• Phase Contrast Microscopy
• Polarized Microscopy
• Interference Contrast Microscopy
• Cytodiagnostic Urinalysis
• Quantitative and Differential Counts
 Red blood cells

• appear as pale discs

• can be confused
with yeast cells
• yeast cells do not
stain and are not
lysed by the addition
of acetic acid
 Dysmorphic RBCs

• Increased numbers in conjunction with RBC cast

bleeding assumed to be renal in origin
• Absence of casts and protein - bleeding assumed
to be non-renal
DRBC
 Leukocytes

• Increased numbers
are seen:
– renal diseases
– urinary tract infection
• when accompanied by
casts:
– renal in origin
 Squamous
Epithelial cells

• Line the distal 1/3 of

the urethra
• Large numbers in
women maybe a source
of contamination
 Transitional Epithelial
cells

• Line the urinary tract

from the renal pelvis
to the proximal 2/3 of
the urethra
• few are present in
normal urine
 Renal Tubular
Epithelial cells

• Small numbers maybe

seen in normal urine
– sloughing of aging cells
• Increased numbers
are seen:
– acute tubular necrosis
– certain drug or heavy
metal toxicity
 Casts

• Formed when an increased numbers of

proteins enter the tubules.
• Formation increases with:
– lower pH
– increased ionic concentration
• Tamm-Horsfall (TH) protein forms the
matrix of all casts
– glycoprotein secreted by cells in the
ascending loop of Henle
 Casts - Some Definitions

• 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:
– Pyelonephritis, chronic
renal disease
– transiently with exercise
– May be a normal finding
 Waxy cast

• Associated with
tubular inflammation
and degeneration
• observed frequently
with chronic renal
failure
 Granular casts

• Appear with glomerular

and tubular diseases
• accompany:
– pyelonephritis
– viral infections
– chronic lead poisoning
 Fatty casts

• Commonly seen
when there is heavy
proteinuria
• a feature of
nephrotic syndrome
• hypothyroidism
 Red Blood Cell casts

• Diagnostic of
glomerular disease
• glomerular damage
allows RBCs to
escape into the
tubules
 White Blood Cell casts

• WBCs enter the

tubular lumen through
and between tubular
epithelial cells
• associated with
pyelonephritis and
tubulointerstitial
disease
 Epithelial Cell casts

• To differentiate from leukocyte cast, supravital

staining and phase -contrast microscopy are
helpful.
• Associated with: tubular necrosis, viral disease
(CMV), heavy metal ingestion
 Bacteria, Fungi, and
Parasites

Bacteria Yeast

Trichomonas Yeast
Clue Cells
 Crystals

• Limited clinical significance

• phosphates, urates, and oxalates are
common and occur in normal urine
• Alkalization and refrigeration promotes
crystals formation
• few crystals are important:
Inherited metabolic disorders
– cystine
– tyrosine
– bilirubin - hepatic and biliary tract diseases
– cholesterol - nephrotic syndrome
 Crystals

• few crystals are important:

– Cystine
– Tyrosine Inherited metabolic disorders
– Leucine
– bilirubin - hepatic and biliary tract diseases
– cholesterol - nephrotic syndrome
 Crystals found in
Normal Urine

Amorphous urates/phosphates Calcium oxalates

Uric acid Triple phosphates

 Crystals found in
Abnormal Urine

Cystine Bilirubin

Tyrosine Cholesterol
 Procedure for Urine
Microscopy

sample centrifugation decantation

collection

Report

slide preparation
writing report microscopy
 Quantitative ME

Issue 1: To centrifuge or not to centrifuge?

“After a 5 min centrifugation of the urine at
3500rpm, only 48% of RBC and 40% of
WBC found to be present could still be
detected under the microscope”
Gadeholt, Brit Med J,1964, 1.1547
 Quantitative ME

Issue 2: To report in /µl or /LPF, /HPF ?

Issue 3: To stain or not to stain?

JCCLS / NCCLS / ECLM Methods
Centrifugation J CCLS NCCLS ECLM
RCF 400 G 500 G 400 G
Time 5 min 5 min 5 min
Urine volume 10 mL S.V.W.I. 10 or 12 mL
Sediment vol. 0.2 mL S.V.W.I. 0.5 or 0.6 mL
Discard aspiration After aspiration
supernatant metering

S.V.W.I. - Standardized Volume within the Institute

JCCLS / NCCLS /ECLM Methods

Centrifugation:

RCF (g) = 1.118 x 10-5 x radius in cm x RPM2

or RPM = 1000 x ( RCF )1/2

11.18 x R

eg. R = 20 cm, speed = 1500 rpm

R = 16 cm, speed = 1700 rpm
R = 10 cm, speed = 2100 rpm

Swing type, balanced, sample should stop naturally

JCCLS / NCCLS / ECLM Methods
Slide J CCLS NCCLS ECLM
Preparation
Staining If needed If needed P.C.- no
(SM stain) B.F.- S stain
Sediment 15 uL Specific Known
volume volume Volume
Coverslip 18 x 18 Standard Defined
mm size size
P.C. - Phase contrast microscope
B.F. - Brightfield microscope
* NCCLS recommends standardized commercial system
JCCLS / NCCLS / ECLM Methods
Microscopy J CCLS NCCLS ECLM
microscope brightfield brightfield phase
contrast
eyepiece x 10 determined x 10–12.5
value
view # of 20 determined 18-22
eyepiece number
objective LP: x 10 determined LP: x 10-16
HP: x 40 value HP: x 40
Examined LPF whole field determined whole field
# of fields
examined HPF at least 10 determined at least 10
# of fields
unit /LPF/HPF /mL /uL, /mL,/LPF
/HPF
 Neubauer counting chamber

3 mm

1 mm W W

Depth of chamber
= 0.1mm

W W
 Fuchs-Rosenthal counting chamber

Small square
Medium square

Large square
1 medium square
= 0.2 uL
16 middle square
Depth of chamber
= 0.2 mm 1 large square
= 3.2 uL

1 mm

1 mm
 Fuch Rose nth al vs Neubauer
fo r Qu antita tive Urin e
Mic roscopy
• Advantage of Fuch Rosenthal over Neubauer
– analysis volume of Neubauer is half of Fuch
Rosenthal
• concentration of urine formed elements is very low
compared with those of hematology.
• For urinalysis, the more volume, the better results
– some urine formed elements are large (ie casts),
these might clog the chamber Depth of chamber
• the deeper the depth, the better
Cells /uL = total cells counted / [mm2 counted x 0.1 mm or 0.2mm]

How many mm squares neubauer fuch rosenthal

were counted chamber chamber
 /HPF, /LPF to /uL conversion
• This depends on the following conditions.
– Real View ( Diameter )
– Magnification# ( x10 or x40 )
– Original Urine Volume before Centrifugation
– Sediment Volume after Centrifugation
– Loaded Sediment Volume on the Slide
– Area of Cover Slip
 Urine ME - Commercial
Systems

Features Count- 10 Kova Urisystem

Initial vol 12 ml 12 ml 12 ml

Final vol 0.8 ml 1.0 ml 0.4 ml

Sediment Conc 15 : 1 12 : 1 30 : 1

Vol of urine used 6 µl 6 µl 16 µl

 Urine ME - Commercial
Systems

Features Count- 10 Kova Urisystem

Area of viewing 36 sq mm 32 sq mm 90 sq mm

No of LPF 11 10 28

No of HPF 183 163 459

Coverslip Acrylic Acrylic Glass

Type
 Procedure for ME

Low power microscopy

• Ensure uniform distribution of urine
sediment
• If uneven distribution, make a new
preparation
• Reduce light intensity
• Scan whole area. Note: sediments tend to
gather along sides of cover-slip
 Procedure for ME

High power microscopy

• Examine 20 - 30 fields (optimal)

But not less than 10 fields
 Procedure for ME

Microscopy with staining

Sediment to stain ratio: 4:1
Types of stains
1.Sternheimer-Malbin Stain (SM Stain)
2.Sterheimer Stain (S Stain)
3.0.5% Toluidine Blue
4. Sudan III and Oil Red O
 Procedure for ME
Reporting Format
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
 Procedure for ME
Reporting Format
Casts
- :0
+ : 1 cast/100LPF or 1 cast/WF
++ : 1 cast/LPF or 100 casts/WF
+++ : 10 casts/LPF or 1,000 casts/WF
++++: 100 casts/LPF or 10,000 casts/WF
or 6 casts/HPF
 Procedure for ME
Reporting Format
Bacteria and Yeast
- :0
+/- : scatter in several fields
+ : seen in each foeld
++ : many or scatter in cluster
+++ : numerous
 Procedure for ME
Reporting Format
Crystal and amorphous materials
- :0
+ : 1 ~ 4/HPF
++ : 5 ~ 9/HPF
+++: 10 ~ /HPF