Urine analysis is an integral part of a clinical laboratory. automation techniques in urine biochemistry, their priniciplas and microscopy along with their advantages and disadvantages are outlined.

1. Automation in urine analysis
Varun Kumar Singh

2. • It all started over 2,000 years ago. Many
cultures once regarded urine as a mystical
fluid, and some still do. Its uses have included
wound healing, stimulation of the body’s
defenses, and disease diagnosis.

3. • Hippocrates (approx. 400 BC) -urine characteristics
(odor / color) - altered with different diseases.
• Gilles de Corbeil - related 20 different types of urine to
conditions of the body (differences in sediment and
color).
• FRANCOIS RAYER & EUGENE N. VIGLA - 1837
• “test strips” - Jules Maumene -1850
• By the end of 19 the century all particles identified.
• AUTOMATED urine microscopy 1993

4. Introduction
• Many different diseases display abnormalities in
urine
• Progression /Regression of various lesions can be
monitored with minimal distress to patient
• Systemic d/s ,Endocrine/Metabolic detected
through recognition of abnormal amount of
disease-specific metabolites excreted in urine
• Simple, non invasive, economical investigation

5. • Most labor intensive subspecialties.
• Least automated
• Least favored.
• Traditionally: chemistry targeted.
• Time: suspended for urine microscopy.

6. Why automate...
• Increases productivity
• Labor Savings
• Improvement in TAT
• Supports Lean Management principles
• Better use of staff
• Reduces errors
• Better response to clinician’s and administration’s
concerns
• Improves precision and accuracy of data
• Better compliance to new federal Medicare regulations
• Ease of Use

7. • Out of all of the analyses performed in the
clinical laboratory the urinalysis has one very
distinct advantage: - it’s a non-invasive test

8. Elements of urine analysis
• 2 step process
• Biochemical analysis
• Microscopy

9. Elements of urine analysis
• The Specimen
• Physical Characteristics
- Color, Clarity, Specific Gravity
• Chemical Analysis
- Glucose, Protein, Bilirubin, Urobilinogen, pH, Blood,
Ketones, Nitrite, Leukocytes, and Ascorbic Acid
• Microscopy
- Formed elements (particles), e.g., epithelial cells,
blood cells, crystals, casts, bacteria, sperm, mucus, etc.

10. Sample collection
• 1st morning void.
• Clean catch.
• Midstream.
• Wide mouthed plastic container.
• Prerequisite – Wash genitalia.
• Avoid – During menstruation.
• Time lag b/w collection & analysis MINIMIZED to avoid :
– microbial over growth
– casts & crystals disintegration.
• Ideal - < 2hrs .

12. Options in automation ...
• Dip sticks
• Automated biochemistry
• Automated microscopy

13. Dipsticks
• Micro chemical system permits qualitative and semi
quantitative analysis within a minimum time span by
simple observation
• Clear plastic strips
• Reagent impregnated paper & Absorbent paper
underneath are held in place on a stiff white carrier foil by
fine Nylon Mesh
• Different reagent areas are affixed on the strip
• Different cellulose areas are impregnated with specific
testing chemicals according to test required

14. •Two external interference factors include:-
1. Glue – interferes with the colour reading
2. Ascorbic acid –inhibits the oxidation reaction -
false negative in c/o hematuria and glycosuria
•Solution : -
1. Adhesion by means of nylon mesh
2. Iodated mesh – prevents ascorbic acid effect

16. Different dipsticks
1. Uristix : Glucose , Protein
2. Multistix –SG : PH, Specific gravity, Glucose, Protein,
Ketone, Bilirubin, Blood, Urobilinogen
3. Multistix -10SG : Also Nitrite & Leukocyte
4. Combistix –SG : PH, Specific gravity, Glucose, Protein
5. Keto –DIASTIX : Ketone, Glucose

18. Advantages of dip stick automation
1. Enhances work flow saving labour and time
2. Standardizes some aspects of manual urinalysis
3. Reduces subjective errors
4. Large number of samples in short time
5. Performed on UNCENTRIFUGED urine

19. Chemical examination using reagent strip
Requirements:
1. Uncentrifuged, fresh, well mixed urine
2. Reagent strips
Procedure:
Dip the test area in urine
Remove excess of urine
Compare test areas with corresponding color
charts, at times specified in good light

20. pH
Principle : Color of reaction area changes depending on PH ,based on
double indicator principle
INDICATORS :
 Methyl red (PH- 4.4-6.2)
 Orange red yellow
 Bromothymol blue(PH-8.0-9.6)
 Yellow blue

21. Influencing
factors
• Nutrition :animal protein-
acidic urine, vegetarian
diet - alkaline urine.
• Metabolic status,diseases
,medicines
Limitations
• stands for too long,
alkaline pH values are
diagnostically
meaningless - bacterial
decomposition
• Residues of disinfectants
(quaternary ammonium
compounds) -false results

22. Principle : Based on Pka change of pretreated polyelectrolytes in relation to
ionic concentration of urine. Indicator substance changes color relative to
ionic concentration ,this is translated to specific gravity values
Deep blue green
Yellow orange
Specific Gravity

23. • Explains differences between microscopy
and test strip results: WBC and RBC - lysed in
low concentrated urine
• Interpretation of borderline results of test
strip parameters: dilution or concentration
of the urine can confirm or invalidate the
pathological significance

24. Limitations
• does not indicate the
contribution
of non-ionic urinary constituents-
urea, creatinine or glucose
• pH >7.0, specific gravity test
strip reading may be too low
and has therefore to be
increased by 0.005 g/mL
• protein -100 to 500 mg/dL or
ketoacidosis, - elevated
• glucose concentrations >1,000
mg/dL (>56 mmol/L) is not
determined
Influencing factors
•Fluid intake
•Heavy sweating
•Increased urine output -
diuretics

25. Glucose
Principle:
Specific GLUCOSE-OXIDASE and PEROXIDASE, a double sequential
enzyme reaction.
Glucose + O2 Gluconic acid +H2O2
H2O2 + Chromogen H20 + Oxidized chromogen changes
REAGENT CLINISTIX MULTI
STIX
CHEM
STRIP
CHROMOGEN O- toluidine KI chromogen Amino
propylcarbazol
Color change Pink blue Blue brown Yellow
Orange brown
Time --- 30 sec 60 sec

26. Limitations
• The urine glucose concentration -glucose excretion - does not necessarily
correlate with the actual blood glucose value
Influencing factors
Low or false-negative glucose
• Metabolic products and drug metabolites which have a reducing action
False-positive glucose
• Presence of residues of peroxide-containing or other strongly oxidizing cleaning
agents

27. Bilirubin
Principle: Based on coupling reaction of bilirubin with a diazonium reagent.
Reagent
strip
Chemstix Multistix
Reagent 2,6 dichlorobenzene
diazonium
tetrafluoroborate
2,4-
dichloroaniline
Time 30—60 sec 20 sec
Result Pink
Violet
Creambuff
Tan

28. Limitations
• High ascorbic acid concentrations lower the sensitivity of the bilirubin test
Influencing factors
False-negative bilirubin
• Prolonged standing - direct sunlight, -oxidation
False-positive bilirubin
• Medicines that color the urine red or that are themselves red in an acid
medium, e.g. phenazopyridine
• Yellow or green reaction color of the UBG test in the presence of high bilirubin
concentrations

29. Urobilinogen
Principle: MODIFIED EHRLICH ALDEHYDE reaction :
Acidic medium
Urobilinogen + chromogen Red colour
REAGENT STRIP MULTISTIX CHEMSTIX
Reagent P-dimethyl amino
benzaldehyde & Acid buffer
4-methoxy benzene-
diazonium
tetrafluoroborate
Time ---- 10-30 sec
Result yellow -red brown Red azo dye
Advantage specific
0.2-1mg/dl
Specific
0.4 mg/dl

30. Limitations
• specific for urobilinogen - not react with other diazo-positive substances
(porphobilinogen, indican, p – aminosalicylic acid, sulfonamides, sulfonylureas)
Influencing factors
False-negative urobilinogen
• Oxidation - in direct sunlight.
• Formaldehyde > 200 mg/dL - preservative
False-positive urobilinogen
• Drugs or metabolites which turn red in an acid medium
(e.g.phenazopyridine)

31. Principle: liberation of oxygen in the reagent strip by peroxidase like
activity of heme from free Hb , lysed Red cell or Myoglobin leading to
oxidation of Chromogen and change in color
Blood
 Dipsticks capable of detecting intact RBC, Free Hb
and Myoglobin
 Lowest detectable concentration is 5 INTACT
RBC/UL or Free Hb to 10RBC/UL

32. REAGENT STRIP CHEMSTIX MULTISTIX
Chromogen &
peroxidase
2,5 dimethyl –2,5
dihydro peroxyhexane
& tetramethyl
benzidine peroxidase
3,3’,5,5’tetramethyl
benzidine & cumene
hydroperoxide
Time 60 sec 40 sec
Results Yellow green Orange green
dark blue
Method of estimation

33. Discrepancy between test and microscopy
• Old specimens- RBCs lyzed in urine upon
sitting-not detected under microscope
• Urine not swirled, RBCs - bottom, pad at the
end of strip being dipped in a concentrated
area
• Over-centrifugation can cause destruction
of RBCs

34. Influencing factors
False-positive blood
• Expired, contaminated or improperly stored strips.
• Residues from strong oxidizing reagents in urine containers or
cleansing tissues
• Menstrual contamination,
• not collecting clean catch midstream
False-negative blood
• Formalin (used as a preservative)
• Nitrite (in excess of 10mg/dL) delays the reaction

35. Principle:
Based PH INDICATORS i.e. proteins carry a charge at physiologic pH, their
presence will elicit a pH change
Reagent strip : Tetra bromophenol blue
Time : 30—60 sec
Protein
Interpretation:
 Yellow Blue
 5-20 mg/dl Albumin can be detected
 Reagent strip more sensitive to albumin than to Globulin,
Bence Jones proteins & mucoproteins

36. Limitations
• Microalbuminuria cannot be detected - first positive result -15–30 mg/dL
• The sensitivity to other proteins (e.g. globulins, proteases, peptones,
mucoproteins) is lower
Influencing factors
Low or false-negative protein
• Proteinuria is mainly consisting out of other proteins than albumin
False-positive protein
• During or after infusion of poly vinyl pyrrolidone
(blood substitute)
• Strongly basic urine (pH > 9) during therapy
with phenazopyridine
• Residues of disinfectants based on quaternary
ammonium compounds or chlorhexidine

37. Principle : Nitroprusside reaction for ketones
Sodium Nitroferricyanide & Glycine+Acetone/Acetoacetic acid
Ketones
Alkaline Medium
Violet colour
REAGENT STRIP CHEMSTIX MULTISTIX
Reagent Sod nitroferricyanide & Glycine Sodium nitroferricyanide & Buffers
Reaction Acetoacetic acid & Acetone Acetoacetic acid
Color
Time
Beige Violet
60 sec
Pink Maroon
15 sec
Results Acetoacetic acid
10mg/dl,acetone70mg/dl
Acetoacetic acid-5-10mg/dl
No reaction

38. Limitations
• Phenylketone or phthaleine compounds - (red-orange )
Influencing factors
False-positive ketones
• Captopril, MESNA (2-mercapto-ethanesulfonic- acid sodium salt) and other
substances containing sulfhydryl

39. NITRITE
Test principle: - The aromatic amine sulfanilamide reacts with nitrite in the
presence of an acid buffer to form a diazonium compound, which is
coupled with 3-hydroxy-1,2,3,4- tetrahydrobenzo-(h)-quinoline to form an
azo dye. Nitrate that is present in the urine is converted by bacterial
reduction into nitrite.
Sulfanilamide + Nitrite Diazonium salt
Diazonium salt + Coupling component  Azo dye (red)

40. Limitations
• The intensity of the red color is
a measure of the nitrite
concentration but cannot be
correlated to the severity of
the infection
Influencing factors
False-positive nitrites
• Expired, contaminated or improperly
stored
Strips
• Drugs that color the urine red e.g.
phenazopyridine
• Bacterial contamination from sample
collection - nitrate to nitrite in
specimens >4 hours old
False-negative nitrites
• Bacteria causing UTIs may not be able to
convert nitrate to nitrite
• Antibiotic therapy
• Insufficient nitrate intake or too short
retention of urine in the bladder

41. Leukocytes
• Test principle :- The leukocytes excreted in the
urine are almost exclusively granulocytes,
whose esterase activity is detected in the test
strip reaction. The test zone contains an
indoxyl ester, which is cleaved by the
granulocyte esterase. The free indoxyl reacts
with a diazonium salt to form a violet dye.

42. Limitations
• The test does not react to
pathogenic bacteria and
trichomonads in urine
• Protein excretion in excess of 500
mg/dL and glucose excretion of
over 2 g/dL could - weaker color
development
Influencing factors
False-positive leukocytes:
• contamination by vaginal secretion
• Expired, contaminated or
improperly stored strips
• Nitrofurantoin, imipenem,
meropenem, clavulanic acid
(antibiotics)
False-negative leukocytes:
• Specimen not mixed well or at a
low temperature
• Proteinuria > 500 mg/dL
• Glucosuria > 2,000 mg/dL
• Cephalexin, gentamycin
• Boric acid, sodium azide, mercury
salts, hydrochloric acid

43. Limitations of dip sticks
1. Differences in lightning conditions
2. Difference in individual skill, failure to keep
specified time
3. Loss of reagent reactivity due to improper
storage
4. Discoloration of strips by bilirubin, blood or
other constituents

44. Role of Quality control in Dip sticks
If tests results are questionable/ inconsistent with expected findings &
clinical history, steps recommended
1. Confirm product is within expiry date
2. Retest with fresh sample
3. Check performance against known Negative & Positive control
materials
4. Check for False positive & False negative

45. REFLECTANCE
SPECTROPHOTOMETERY

46. • Instruments intended for single use
• Semi automated urine analysis systems
• Fully automated urine analysis systems

47. •Principle : REFLECTANCE SPECTROPHOTOMETERY
• Analyses color and intensity of light reflected from reagent area and reports
results in clinically meaningful units
• NO calculations required
• Automatic calibration : Runs a self test each time before each strip is read or
power is switched on.
URI PLUS 1A

48. Method of Operation
• Strips laid on the instrument
• Sensor detects strip presence and
activates strip movement ,reading cycle
• Has an optional Bar code reader.
• QC done once in morning

49. UriPlus 900
• Fully automatic
• 10 & 11 parameter Strips used
• Based on Reflectance photometery
{colorimetry}
• Uses high lumonosity 4 wavelength
cold light source reflection
determination technology.

50. Calibration

51. Quality control
Once a day in morning

52. How tests are done in our lab?

54. Cobas 6000 c501

55. Electrochemiluminiscence

56. Can analyse urine :
 Mg+ , Na+ ,K+ , Ca++, P-
 Protein
 Microalbumin
 Uric acid
 Urea
 Creatinine
 Amylase
 Micro total protein
•Light emitting substance like ruthium
labelled Ab are added to the sample.
•Emission of light is electrically
stimulated
•Amount of light produced is directly
proportional to amount of substance to
be detected is present
Cobas 6000 c501
Based on Electrochemiluminiscence (ECL) technology:

57. Urine microscopy
• Bright light microscopy
• Phase contrast microscopy
• epithelial cells, blood cells, crystals, casts,
bacteria, sperm, mucus, etc.

58. Automation in microscopy
Flowcytometry Auto particle recognition
SYSMEX UF-
50, 100,
1000i
IRIS Q 200
URI SED
AVE-763Analyzer

59. Flowcytometry
•Particles - labeled with fluorophores
•measured in a laser beam
•classified
•fluorescence
•size
•impedance
•forward scattered light.
•results - scattergrams and histograms.
•cells per microliter or cells per field of view

60. Auto particle recognition
•Particle images - planar flow cell in the
object plane of a microscope.
•Stroboscopic illumination freezes the motion - blur-free
images - charge-coupled device camera sensor.
•Individual particle images isolated from each of the 500
captured frames - 12 categories
• their size, shape, contrast and structure
•The images - verification and manual editing
•Results - particles per field of view
(per high-power field; HPF) or per microliter.

61. Kova systems
• Semi automated
• Kova tube (12ml), kova
cap
• Centrifuge 1500rpm x 5
min
• Kova petter - decant,
resuspend, charge
• Kova slide - counts

62. Closed slide system
• Censlide
• Fast centrifuge <2 min, 1350rpm
• Standarised sediment volume
• Transfer to scope

63. Particle count algorithm/ volume calibration = particle concentration
User defined
criterion met
yes
no
LIS
Flags for manual
confirmation
Manual
microscopy

68. ADVANTAGES DISADVANTAGES
• Important screening tool;
• Reduces work load in a busy
laboratory - reducing the samples
to be examined manually.
• Not affected by Preanalytic
confounding factors like improper
centrifugation
• Excellent walk away efficiency.
• Fast results on large no.of samples.
• Accurate no. given for most
particles.
• Review of sample possible.
• DOES NOT SUBCLASSIFY the
particle.
• NO INTERNATIONAL
STANDERDISATION or recognized
REFERANCE MEASUREMENT
procedure drawback in result
validation.
• Insufficient mixing leads to wrong
analysis.
• Cells like Dysmorphic WBC
mistaken as artefact

69. COMPARISION WITH MANUAL
SN Variable Manual Automation
1. Bias ++ Nil
2. Standardisation + Absent
3. Precision + / - ++
4. Reproducability + / - ++
5. Variance ++ Nil
6. Crystal,cast,&
microbial sub
categorization
Excellent Absent
7. Quantitation rbc
wbc
Estimate Exact No.
8. Time More Less
9. Cost Effective Expensive

70. Summary
• precise and improves the work flow in a routine laboratory
• Rapid biochemical analysis
• sediment analysis – helps in identifying pathological samples -
missed in the two-step procedure.
• combined with dipstick testing- reduce the number of
specimens submitted to microscopy.
• Visual microscopy - dysmorphic erythrocytes, yeasts,
Trichomonas, oval fat bodies differentiation of casts and
certain crystals.

71. References
• Henry’s clinical diagnosis and management by laboratory methods,
22nd edition
• Jeff A. et al, Urinalysis: A Comprehensive Review, American Family
Physician, 71,6
• Compendium of urinalysis: Urine test strips and microscopy, Cobas
• Nousin et al, Automated urinalysis: first experiences and a
comparison between the Iris iQ200 urine microscopy system, the
Sysmex UF-100 flow cytometer and manual microscopic particle
counting, Clin Chem Lab Med 2007;45(9):1251–1256
• Automating urinalysis , Iris diagnostics
• Langlouis et al, Automated Flow Cytometry Compared with an
Automated Dipstick Reader for Urinalysis, Clinical Chemistry
45:1,118–122 (1999)