Sefehrhtbf

1. AUTOMATION IN URINE ANALYSIS
Presented by: Swati Mishra
Moderator : Dr P. Kusaraju (Asst. Prof)

2. CONTENTS
• Introduction
• Indications
• Elements of urinalysis
• Sample collection
• Automation
• Comparison of automated analyzers
• Comparison of manual and automated urinalysis
• Summary

3. Introduction
• Urine analysis is one of the most commonly
performed laboratory tests in clinical practice.
• Microscopic examination of urine is also called as the
‘liquid biopsy of the urinary tract’.

4. Indications
• Suspected renal diseases - glomerulonephritis,
nephrotic syndrome, pyelonephritis, and renal
failure.
• Urinary tract infection
• Metabolic disorders like diabetes mellitus
• Differential diagnosis of jaundice
• Plasma cell dyscrasia.
• Diagnosis of pregnancy.

5. Elements of urinalysis
• Physical examination – Volume , specific
gravity, odor, appearance/clarity, color
• Chemical examination – pH, proteins, glucose,
ketones, bilirubin, bile salts, urobilinogen,
blood, hemoglobin, myoglobin,
nitrite/leucocyte esterase
• Microscopy – cells, casts, crystals, organisms

6. Sample Collection
• First morning void, midstream, clean catch
• Wide mouthed plastic container
• Time lag between collection and analysis
should be avoided

8. Automation in Urinalysis
• Manual microscopic urine sediment examination is
labor intensive, time consuming and lacks
standardization in high volume laboratories.
• Goal of automation – maximize productivity and
testing quality, while keeping expenditures and
turnaround time minimum.

9. Several automated instruments are currently available
to standardize:
• Sample processing
• Biochemical test strips analysis
• Microscopy analysis
• Report results

10. Automated instruments in urinalysis include
• Reagent test strips/Dipsticks: performed manually, or
by
Semi-automated chemistry analyzers: test strip
dipped in urine manually and analyzed by an
instrument
Fully automated chemistry analyzers: add urine to
the reagent strip and process.

11. • Automated urine cell analyzers : mix, aspirate,
dilute, and stain urine to classify urine
sediment particles.
• Automated urine systems: perform a complete
urinalysis that includes the physical, chemical,
and microscopic parts of a routine urinalysis.

12. Reagent test strips/Dipsticks
• Microchemical system
• Qualitative and semi-quantitative analysis within
minimum duration
• Reagent impregnated paper and absorbent
paper held in place by fine nylon mesh
• Different reagent areas (cellulose areas
impregnated with specific testing chemicals)
affixed on strip

14. Uses of Dipsticks

15. Different dipsticks
1. Uristix : Glucose, Proteins
2. Multistix-SG : pH, specific gravity, glucose, proteins,
ketone, bilirubin, blood, urobilinogen
3. Multistix-10SG : +nitrite & leucocyte
4. Combistix –SG : pH, specific gravity, glucose,
proteins
5. Keto-DIASTIX : Ketone , glucose

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

18. pH
• Principle- Test area contains polyionic nylon polymer
bound to Hydrogen ions, which is released on
reaction with cations in urine causing change in color
of the pH-sensitive dye.

19. Proteins
• The reagent area of the strip is coated with an
indicator and buffered to an acid pH which changes
color in the presence of proteins. The principle is
known as “protein error of indicators”.

20. Glucose
• Based on glucose oxidase-peroxidase reaction.
Reagent area of the strips is impregnated with two
enzymes (glucose oxidase and peroxidase) and a
chromogen.

21. Blood
•Lowest detectable
concentration – 5 intact RBCs

22. • Ketones :
• Bilirubin : based on reaction of with diazo reagent;
color change is proportional to the concentration of
bilirubin.
• Urobilinogen : area is impregnated with either p-
dimethylaminobenzaldehyde or 4-methoxybenzene
diazonium tetrafluoroborate.

23. • If tests results are questionable/ inconsistent with expected
findings & clinical history, steps recommended are-
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

24. Limitations of dip sticks
• Differences in daylight conditions
• Difference in individual skill, failure to keep specified
time
• Loss of reagent reactivity due to improper storage
• Discoloration of strips by bilirubin, blood or other
constituents

25. Automated chemistry analyzers
• Working principle – reflectance spectrophotometry
1. URI PLUS 1A:
 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.

26. Method of Operation: Strips laid on the instrument
Sensor detects strip presence and activates strip
movement, reading cycle
Has an optional Bar code
reader.
Quality control done
once in morning.

27. UriPlus 900
• Fully automatic
• 10 & 11 parameter strips used
• Based on
Reflectance photometery
• Uses high luminosity
4 wavelength cold light source reflection determination
technology.

28. Manufacturers use one of three technologies to
perform automated urine microscopic analysis:
• Digital flow microscopy,
• Flow cytometry
• Cuvette-based digital microscopy

29. Principle Machine
Digital flow microscopy Iris iQ200 microscopic
analyser, URISED
Flow cytometry UF-1000i, UF-100 analyser,
AUTION HYBRID
Cuvette-based digital
microscopy 77 Elektronika

30. Iris iQ200 microscopic analyser
• The machine for Iris Diagnostics USA uses digital
imaging and auto particle recognition to classify and
quantify urine particles in uncentrifuged urine based
on size and shape.
• Works on the principle of digital flow microscopy.

31. • Automatically analyzes and classifies urine particles
into 12 categories.
• The sample is mixed and aspirated to a planar flow
cell where 500 digital photomicroscopic images are
taken per sample.
• This system uses Auto particle recognition (APR)
software.

33. Flow cytometry –Sysmex UF-1000i
• Particles labeled with fluorophores/fluorochromes
(Polymethine dye).
• Flow cytometric analyzers identify and categorize
particles in urine based on :
forward scatter
fluorescence staining characteristics
impedance signals
adaptive cluster analysis
side scatter (specific for bacteria).

34. • Identification of particles based on – size,
shape, volume and staining characteristics
• Separate channel for bacterial analysis
(improves bacterial detection)
• Sample volume : 4-5ml (1ml in manual mode)
• Results (scattergrams & histograms) – in
cells/microL

35. Urine sample aspirated into analyzer
Divided to 2 channels for sediment and bacterial analysis
Staining with fluorescent dye and dilution
Particles oriented to single file by flow cell and
hydrodynamic focusing
Analysis by single red semiconductor laser (λ635nm)
Urine particle categorization
Results (scattergram)

37. UF-1000i Particle detection categories
Particles enumerated Flagged particles
RBCs Non hyaline (pathologic)
casts*
WBCs Crystals*
Epithelial casts Small round cells*
Hyaline casts Yeast
Bacteria Mucus
Sperm
*Manual microscopic examination required to specifically
identify and categorize

38. • Manual microscopic examination to:
Classify Non hyaline casts (granular, cellular, RBC,
WBC, Crystalline)
Identify crystals
Identify yeast
Categorize particles identified as small round cells
as transitional cells, renal cells or another small
particle.

39. A. Forward scatter v/s
fluorescent light intensity –
high sensitivity
B. Forward scatter v/s
fluorescent light intensity –
low sensitivity

40. • Results from UF-1000i can be electronically linked to
urine chemistry analyzer  Integrated urinalysis
report
• AUTION HYBRID : integrated analyzer (chemistry and
microscopy analyzers within single unit)
• User defined criteria adjusted to reduce review rate
of specimens and increase productivity.

41. Cuvette-based Particle analysis
• Recent innovation
• Urine specimen centrifuged in a cuvatte
• multiple images captured by high resolution digital
camera coupled to a microscope
• Sedimax /UriSed & Cobas U 701 are the only
cuvette based urine analyzers

44. • The UriSed 2 microscopic urine sediment analyzer is a stand-alone
instrument which can be connected to the LabUMat 2 urine strip
reader.
• Together, the two instruments make a Complete Urine Laboratory
System.

45. Technical specifications of the 3 machines
iQ 200 Sprint UF 1000i UriSed
Technologies Flow cell digital
imaging with
automatic particle
recognition software
Fluorescence flow
cytometry with diode
laser and
hydrodynamic
focussing
conductometry
Microscopic urine
sediment analysis,
digital imaging,
automatic particle
recognition.
Samples/hour 101 Normal mode – 100;
special mode - 80
80
Sample volume 2ml 4ml (1ml in manual
mode)
0.2 ml
Data storage 10000 pt results with
images
10000 samples
graphics, 5000 patie
nt info, 1000
selective test orders.
50000 sample results
and images
Size (mm) and weight
(kg)
559x610x530 ; 46kg 615x710x580; 75.5kg 600x600x600; 58kg

46. Comparisons between different automation machines for urinalysis
SYSMEX UF 1000i Iris iQ 200 UriSed
Advanta
ges
1. Improved bacteria
counting and flags
for UTI
2. Uses uncentrifuged
sample
3. Manual operation
for urgent testing.
1. uncentrifuged
samples.
2. Accurately
quantifies
bacteria.
3. Results may be
viewed from
archived images
1. No reagents
required
2. Results viewed from
digital images.
3. Images same as
manual microscopy
so minimal
retraining needed
Disadva
ntages
1. Pathologic casts
not differentiated
from benign casts
2. Flagged results
require manual
microscopy
1. Technical training
to view images.
2. Mistakes crystals
for RBC and
cannot classify
dysmorphic RBCs
1. Ultracentrifugation
causes particle loss
2. Mistakes yeast for
rbc
3. Mistakes EC for
WBC and not FDA
approved

47. Comparison with Manual
SN Variable Manual Automation
1 Bias ++ Nil
2 Standardisation + Absent
3 Precision +/- ++
4 Reproducability +/- ++
5 Variance ++ Nil
6 Crystal, cast,
microbial
subcategorization
Excellent Absent
7 Quantitation of
RBCs & WBCs
Estimate Exact no
8 Time More Less
9 Cost Effective Expensive

48. Summary
• So a complete urinalysis using macroscopic examination,
reagent strips , automation and manual microscopy can be
used in conjunction for standard and accurate results.
• Automation has more benefit in setups with high sample
load.
• Automation helps to screen samples for UTI or kidney
disease.

49. REFERENCES
• Henry’s clinical diagnosis and management by
laboratory methods, 24th edition
• Nancy A. Brunzel - Fundamentals of urine and
body fluid analysis, 4th
edition - chapter 16
• Shirish M. Kawthalkar -Essentials of clinical
pathology

50. THANK YOU