AUTOMATION IN URINALYSIS

By P. DINESH
1st year PG
Moderator
Dr. N. Bharath Rao MD,DCP
Professor and HOD
Department of Pathology
OUTLINE:
• Introduction.
• Indications of urinalysis.
• Collection of urine.
• Urine transport and preservation.
• Urinalysis instrumentation.
• Siemans Clinitek Status+ analyser.
• Manual urine sediment analysis.
• CUVETTE-BASED MICROSCOPY: UriSed/sediMAX.
• Sysmex UF-1000i.
UF-1000i Reagents.
UF-1000i Sample Volumes.
• IQ Automated Urine microscopy analyser.
• Advantages.
• Disadvatages.
Introduction
:
• Urinalysis is one of the most commonly performed laboratory test
in clinical practice.
• Manual urine testing remains the most widely used method for
assessment of urine sediment. However, automated urine testing
protocols are not in consensus.
• In the manual urinalysis technique, microscopic examination is
used to check for the presence of formed elements. Although the
centrifugation step to concentrate the sample is necessary, it remains
a major source of errors, as it leads to a variable loss of erythrocytes
and leukocytes.
Cont…
• Strict standardization of all procedures for collection, transport,
sample preparation, and analysis are mandatory as the basis of an
effective diagnostic strategy for urine analysis.
• Different automated analyzers are currently used to perform the task
of automated urine analysis.
• These analyzers adopt two methodologies for urine sediment
analysis,
1. Electrical impedance.
2. Image-based analysis systems that sort particles according to
predetermined particle dimensions
INDICATIONS OF URINALYSIS:
• Suspected renal diseases like glomerulonephritis, nephrotic syndrome,
pyelonephritis, and renal failure.
• Detection of urinary tract infection.
• Detection and management of metabolic disorders like diabetes
melitus.
• Differential diagnosis of jaundice.
• Detection and management of plasma cell dyscrasias.
• Diagnosis of pregnancy.
COLLECTION OF
URINE:
• Time of collection:
1. A single specimen: This may be a first morning voiding, a random
specimen, or a post prandial specimen.
The first voided specimen in the morning:
The most concentrated and has acidic pH formed elements(cells and
casts) are well preserved.
• Routine examination.
• Microscopic analysis of cellular elements.
• Bacteriological analysis.
• For glucose, protein and nitrite.
• The random specimen:
For routine urine examination.
• Post-prandial specimen:
For estimation of glucose or of urobilinogen.
2. 24 hour specimen:
After getting up in the morning, the first urine is discarded.

This method is used for quantitative estimation of proteins and

hormones.
Urine transport:
• An increased time lag between sampling and analysis, a lack of
temperature control and a lack of addition of a preservative to samples for
which urinalysis cannot be performed within two hours of collection, will
lower the quality of urinary test results.
• A sterile collection is mandatory for bacteriological culture of urine
samples. 
• The original primary sample should be divided into different smaller
aliquots for morphological, microbiological and chemical analyses to
decrease the risk of contamination.
• Easy filling from primary containers without risk of spillage should be
one of the characteristic of secondary containers. These transparent tubes
preferably have a round bottom, which allow a better resuspension of the
sediment following centrifugation.
Cont…
• Vacuum systems have been developed, allowing direct sample
aspiration into a secondary container.
• All urine collection and/or transport containers should be clean and
free of particles or interfering substances.
• The collection and/or transport container should have a secure lid,
non reusable and be leak-resistant.
• It is good practice to use containers that are made of break-resistant
plastic, which is safer than glass.
Urine preservation:
• NCCLS guidelines for microbiological urine testing recommend the
use of chemical preservatives if the specimen cannot be processed
within 2 hours of collection. Otherwise, these specimens should be
refrigerated at 2-8°C.
• Alkaline pH, low relative density and low osmolality can induce a
rapid lysis of some urine particles after collection.
• Addition of stabilizers usually prevents metabolic changes of urine
analytes and overgrowth of bacteria.
• Traditionally, ethanol (50%) is selected to preserve cellular
particles, although only a partly prevented lysis of red and white blood
cells is observed.
• Addition of polyethylene glycol (20 g/L) to the ethanol fixative
(Saccomanno’s fixative) improves preservation quality (10,25).
• Lyophilised formulations should be chosen among the commercial
preservatives as there is no risk of sample dilution of spillage.
• Also, containers supplemented with boric acid alone or in
combination with formic acid or other stabilizing media, are used 
Urinalysis Instrumentation:
• The biggest preventable variable in urinalysis results comes from the
mis-reading/mis-interpreting of the dipstick results. The solution to this
problem is utilization of an automated urine dipstick reading instrument.
• Inside the urinalysis dipstick reader, a filtered focused light is directed
to the dipstick pad where, depending on the depth of pad’s reacted color,
some of it is absorbed and the remainder is bounced/reflected to a
photocell - detector.
• The signal created in the photocell detector is sent to the instrument’s
on-board computer which compares the amount of reflected light
detected to that of known concentrations of the analyte/substance being
measured and displays the appropriate concentration for the amount of
light detected from this analysis.
• Urinalysis dipstick reading instruments vary greatly in size, cost
and the tasks they perform, but all strip readers evaluate the
reactions on the dipstick at specified times.

• Their accuracy depends on the operator’s skill and accuracy in

appropriately identifying the sample, mixing, dipping, blotting,
getting the strip onto the tray and activating the timer appropriately.

• Higher end instruments add the urine to the strip and

automatically activate the timing mechanism.
 Advantages to use of such instrumentation includes:
• Increased efficiency,
• Improved precision,
• Accuracy and Reproducibility.

• Three ways to perform test strip analysis:

• MANUAL: The test done by hand.
• SEMIAUTOMATED: The test strip dipped in the urine manually and
then analysed by instrument.
• AUTOMATED: The test strip is analysed completely by an instrument.
Siemans Clinitek Status + analyser:
Siemans Clinitek Status + analyser:
• Semi-automated, benchtop instrument designed to read the
Siemans Multistix 10 SG Reagent Strip.
• works on the principle of reflectance photometry.
• Light from the LEDs travels along the light guide and is reflected off
the calibration bar, strip or cassette onto the mirror. It is then directed
through an aperture on the lens, from where it is focused onto the
detector.
• The light intensity detected is converted into electrical impulses,
which are processed by the instrument’s microprocessor and converted
into clinically meaningful results.
• The programming module for the Multistix 10 SG Reagent Strips
contains programming information necessary to read the reagent strip
areas for testing of
• Glucose,
• Bilirubin,
• Ketone (Acetoacetic Acid),
• Blood,
• Ph,
• Protein,
• Urobilinogen,
• Nitrite And Leukocytes.
• The instrument is programmed with such information as
wavelengths, error messages, operating sequence, and algorithms
needed to convert reflectance into clinically meaningful results.
• A white plastic strip located at the end of the feed table insert,
provides a reflectance surface for the internal calibration of the
instrument’s optical system.
• Calibration is accomplished automatically by the measurement of
reflected light from the calibration strip surface. The calibration strip
(aka - the white bar) must be kept clean for proper instrument
calibration.
Microalbumin 2 Reagent Strips:
• Albumin,
• Creatinine, and
• Albumin To Creatinine ratio results in 1 minute
• Can be used by POC or physicians’ offices
• Use with Clinitek 50 or Clinitek Status analyzers

• Sensitivity as low as 2mg/dL for urine protein

• More reliable; less affected by interferences (e.g. specific gravity and
pH)
Microalbumin 9 Reagent Strips:
• Albumin,
• Creatinine,
• Blood,
• Glucose,
• Ketone, Leukocyte, Nitrite,
• Ph, & Protein and Albumin To Creatinine ratio & Protein to creatinine
ratio

• Use with Clinitek Status or Advantis analyzers

• Random sample; no timed or 24 hr urine sample required
• Accurate identification of microalbuminuria
A wide range of test parameters:
Clinitek status is suitable for use with a wide range of multistix tests
including:
• Mutlistix 10SG
• Mutlistix 8SG
• Multistix GP
• Clinitek microalbumin
• Clinitest hCG
Manual Urine Sediment
Analysis:
Analytic variables
• Mixing of samples by inversion, not swirling
• Standardized volume for centrifugation; note volume if less than 12mL
• Time and G force for centrifugation; do not use brake
• Inconsistent decantation and re-suspension steps after centrifugation.
• Reduced recovery rate of urine elements after centrifugation
• Variability in concentration ratio
• Supernatant removal
• Mixing of suspension
• Filling of chamber; technique-dependent
• Distributional errors
Cont…

• Commercial slide systems

• Provide some standardization
• Technique-dependent
• Vary in concentration ratios: 1:5 to 1:48
• Low & high power fields of view are microscope dependent;
reporting unit inequity
Manual Urine Sediment
Microscopy
• Subjective element identification
• Poor reproducibility
• Lack of standardization
• Time consuming/labor intensive
• Three types of instruments are on the market, each one being based
on its own technology:

• Cuvette-based microscopy (UriSed/sediMAX).

• Flow cytometry (UF-1000i, Sysmex)

• Automated intelligent microscopy (iQ200, Beckmann)

CUVETTE-BASED MICROSCOPY:
UriSed/sediMAX
• A walk-away automatic urine sediment analyser.
• It supplies B/W images of particles within whole fields of view.
• These are similar to the microscopic fields seen with manual
microscopy.
• Throughput: 100 samples/hour.
• Particles identified are
• Erythrocytes • Leukocytes • Squamous epithelial cells • Non-squamous
epithelial cells • Hyaline casts • Pathological casts • Crystals: CaOx, UA,
struvite • Bacteria
• A single-use patented cuvette

is filled with automatically mixed native urine.

(volume aspirated: 2.0 mL, volume examined: 2.2 µL)
• The sample is centrifuged within the instrument(10 seconds at 260 g)
• The cuvette is forwarded to the microscope table.
• An automatic focusing at different levels is performed
• A built-in camera takes a digital image of each field of view
(magnification: ~400x)
• For each sample 15 images are taken.
• Identification and quantitation of the particles (as No/µL or No/HPF) is
carried out by Auto Image Evaluation Module (AIEM), a complex
artificial neural network structure which has specifically been
developed for the instrument
• WHOLE FIELD OF VIEW: Many WBCS and WHOLE FIELD OF VIEW: Isomorphic
bacteria RBCs and deep transitional cells
URINARY TRACT INFECTION UROLOGICAL DISEASE
WHOLE FIELD OF VIEW: Dysmorphic RBCs and fatty
particles
NEPHROTIC SEDIMENT
Sysmex UF-1000i
 Sysmex UF-
1000i
• Fully automated sample preparation(mixing, aspirating, diluting,
staining).
• Objective particle characterization and identification based on
detection of forward scatter, fluorescence and on adaptive cluster
analysis.
• Laser-based flow cytometer utilizing 2 stains with fluorescent dyes to
stain cellular elements.
• Separate bacteria channel for improved discrimination.
• Main unit with integrated pneumatic unit
• IPU (information processing unit) Windows XP operating system
• Sampler unit with tube rotator unit
• Bar code reader
• Laser Jet graphic printer/line printer (1 device, 2 settings)
• Handheld bar code reader
UF-1000i Tube Rotator
 UF-1000i Reagents

UFII SHEATH™

UFII PACK™-BAC
UFII PACK™-SED

UFII SEARCH™ -BAC

UFII SEARCH™-SED
 UF II PACK-SED / UF II SEARCH-
SED
• UF II PACK-SED
• Removal of amorphous salts together with heating (up to 35°C)

• UF II SEARCH-SED
• Polymethine dye
• Chromogen chain with electron donor and acceptor group

• Stains parts of nucleus, parts of cytoplasm and membranes

• Excitation wavelength is 635 nm
• Emission wavelength is over 660 nm
 UF II PACK-BAC / UF II
SEARCH-BAC
• UF II PACK-BAC
• UF II PACK-BAC (e.g. its pH value) together with heating to >40°C
suppresses non-specific staining of particles other than bacteria
• UF II SEARCH-BAC
• Polymethine dye
• Distinctively stains nucleic acid elements in bacteria
 UF-1000i Sample Volumes
• Minimum sample volume:
• Manual mode: 1 mL
• Sampler mode: 4 mL
• Aspiration volume:
• Manual mode: 800 µL
• Sampler mode: 1,200 µL
• Processed sample volume (SRV) in sampler and manual mode:
• 150 µL for the sediment analysis
• 62.5 µL for the bacteria analysis
• Throughput Up to 100 samples per hour.
• Sampler 50 samples on board processing.
• Parameters RBC, WBC, Epithelial Cells, Casts, Bacteria
• Flagging Parameters Pathological Casts, Crystals, Small Round Cells,
Sperm, Yeast, Mucus
• Data Storage 10,000 samples (incl. Scattergrams)
• Peripheral Interfaces Host line printer Graphic printer Handheld
barcode reader
• Quality Control 24 files with 300 data points each Sysmex control
material: UF II CONTROL Levey-Jennings and X-bar M quality control
programs
• Accessories Graphic printer
 Sample Volumes and Dilution

• Addition of reagent leads to a dilution of the urine

• for the SED analysis exactly by the factor 4:
• 150 µL sample plus 435 µL diluent plus 15 µL dye equals 600 µL

• for the BAC analysis exactly by the factor 8:

• 62.5 µL sample plus 425 µL diluent plus
12.5 µL stain equals 500 µL
Mechanism:
 Laminar Flow
Scattered
Laser light light

Flow cell

particles
Sheath
reagent
Sheath
nozzle
• The Sysmex UF-100 (TOA Medical Electronics Co.) uses argon laser
flow cytometry.
• The UF-100 aspirates 800 m L of uncentrifuged urine, dilutes the
sample four times to dissolve the crystalline content,
• Measures the urine conductivity, and
• Analyzes the urinary formed elements by electrical impedance for
volume,
• Forward light scatter for size,
• Fluorescent dyes for DNA (phenanthridine) and membranes
(carbocyanine).
• The pulse intensity and width of the forward scattered light
and fluorescence light are measured.
• From these data, together with the impedance data, the
urinary formed elements are categorized by multiparametric
algorithms on the basis of their size, shape, volume, and staining
characteristics.
• The results are displayed in scattergrams, histograms, and in
counts per microliter as well as counts per high-power field (HPF).
UF-1000i Detection Parameters
Enumerated Parameters Flagged Parameters

RBC Pathological Casts

WBC Crystals

Epithelial Cells Small Round Cells

Hyaline Casts Yeast

Bacteria Mucus

Sperm
• The chemstrip superautomated urine analyser and Roche
Diagnostics urisys 2400 system are fully automated ‘walk away’
urine chemistry instruments for a large urinalysis.
• With the Chemstrip super automated urine analyser, sample
volumes are detected, adjusted and automatically mixed.
UF-1000i Technology
Improved
determination of
bacteria
Sediments Bacteria Diluents

Sediments Bacteria
Incubation

Detection
unit
Sediments Bacteria Stain
Red semiconductor laser
•Down sizing
•Long life
Two chambers for stain and dilution
•Reduced power
consumption
UF-1000i
Sediment 1
UF-1000i
Technology
1) Enhanced detection of
bacteria
Dye
Dye
Dye
Dye DyeDye
Dye Dye
DyeDye
Dye
Dye Dye
Dye
DyeDye
DyeDye
Dye
Dye
2) Staining bacteria Dye
Dye Dye
Dye
Dye
Dye
Dye Dye
nuclei
Forward Scatter Non-specific staining with debris

Specific stain for Nucleic Acid

Polymethine dye

　 Stain DNA/RNA

Fluorescence
UF-1000i
Bacteria 3
UF-1000i Sample with No Flagging
UF-1000i Sample with Flagging
 UF-1000i Anti-Carryover Action

• Trigger: bacteria count only

• Sequential mode:
• If the bacteria count exceeds the cut-offs preset in the anti-carry-
over settings, additional autorinse cycles are performed before the
next sample is aspirated.
• Overlapping mode:
• If the bacteria count exceeds the cut-offs preset in the anti-carry-
over settings, additional autorinse cycles are performed. The next
sample will be aspirated twice.
UF-1000i Anti-Carryover
UF-1000i Quality Control
• UF II Control: two level commercial controls containing particles
representing RBC, WBC, EC, casts, and bacteria
• Controls also monitor conductivity plus the high level monitors
sensitivity parameters-FSC, FSCW, FLH, FLL, FLLW, SSC.
• Levy Jennings & Radar Charts
UF-1000i 24 Control Files
 UF-1000i QC
Charts
L-J Charts
Radar Charts
300 data points
 IQ Automated Urine microscopy analyser:
 Automatically analyses and classifies urine particles in to 12
catagories. The sample is mixed and aspirated to a planer flowcell
where 500 digital photomicroscopic images are taken per sample.
 The system uses Auto particle recognition(APR) software that
classifies urine particles in the photographs based on size, shape,
texture and contrast in to 12 categories-
 RBCs, WBCs, WBC clumps, hyaline cast, unclassified cast,
squamous and non squamous epitheilial cells, bacteria, yeast,
crystals, mucus and sperm.
An automated microscope is focalized on a planar flow cell, in which the
particles flow as a sheet, being sandwiched between two layers of an
enveloping fluid
• A stroboscopic lamp, firing 24 bursts/second, stops the motion of the
particles passing through the camera
• The stopped motion view is observed through magnifying lenses
• The images are collected by a videocamera
EXAMPLE OF IMAGES SUPPLIED BY iQ200 (URIC ACID)
Advantages
:
Objective, analytical measurements
New levels of precision and reliability.
Unprecendented convenience.
Time saving simplicity.
Reassuringly proven performance.
Minimal training required.
Decreased labor expense
Audit compliant.
Pregnancy test capability using the new Clinitest hCG.
Automation reduces the risk of errors.
Disadvantages:
References:
1.  Coppens A, Speeckaert M, Delanghe J. The pre-analytical
challenges of routine urinalysis. Acta Clin Belg. 2010;65:182–9. [
PubMed]
2.  Carlson DA, Statland BE. Automated urinalysis. Clin Lab
Med. 1988;8:449–61. [PubMed].
3. NCCLS GP-16A2, Volume 21, No. 19, Urinalysis and Collection,
Transportation, and Preservation of Urine Specimens; Approved
Guideline-Second Edition, p. 4-21.
4. Ramnik sood volume 1, chapter-4, pg no
Cont..
5. Siemens Clinitek Status+ users guide (
http://www.medical.siemens.com/siemens/en_GLOBAL/gg_diag_
FBAs/files/POC/Urinalysis/Clinitek_Status_Bro chure_update_Rev
02.pdf
).
6. Winkel P, Statland BE, Jorgenson J. Urine microscopy: an
illdefinedmethodexaminedbyamultifactorialtechnique.ClinChem
1974;20:436–9.