automated urine analysis
TRANSCRIPT
AUTOMATED URINE ANALYSIS
Presenter : Dr Manjunatha T M Moderator: Dr. Rajashekhar. K.S
The history of urinalysis
The ancient worldThe origin of visual urine diagnostics, can be traced back to ancient Egypt. Hippocrates (approx. 400 BC) recognized that urine characteristics (odor / color) were altered with different diseases.
Six centuries later, Galen (AD 129–200) refined Hippocrates
ideas, theorizing that urine represented is not a filtrate of the
four humors and but rather, a filtrate of the blood . An increasing number of physicians were diagnosing from
urine alone. Amateurs (called‚leches‘) started diagnosing based only
on the color of urine.
The first “test strips” were developed by the
Parisian chemist Jules Maumene (1818–1898)
when, in 1850,
he impregnated a strip of merino wool with “tin
protochloride” (stannous chloride).
On application of a drop of urine and heating over
a candle the strip immediately turned black if the
urine
contained sugar and it took another 70 years before
the Viennese chemist Fritz Feigl (1891–1971)
published his technique of “spot analysis.”
Urine test strips in the sense used today were first
made on industrial scale and offered commercially
in the 1950s.
The company Boehringer Mannheim, today a top
leader on the world market under the name of
Roche,
launched its first Combur- TestR strips in 1964.
Urine analysis is a valuable tool used to diagnose and monitor renal and urinary tract illnesses. Typically it is a moderate to high sample volume test for a general chemistry lab, representing up to 30 % of all samples received .Routine urinalysis consists of macroscopic examination, chemical analysis and microscopic urine sediment examination.
Urinalysis Automation Several automated instruments are currently available to standardize:
• Sample processing• Biochemical test strips analysis• Microscopy analysis• Report results
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Automation Urinalysis Features:
1. On-line computer capability2. Bar coding3. Manual entry of color4. Clarity5. Microscopic results
There are three ways to perform a test strip analysis:
• Manual – The test is done by hand
• Semi-automated – The test strip is dipped in the urine
manually and then analyzed by an instrument
• Fully-automated – The test strip is analyzed
completely
by an instrument
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 Automation Equipment Manufacture
1) Semiautomated Chemistry Instruments
2) Clinitek 200/200 + Siemens Medical Diagnostics3) Clinitek 500 Siemens Medical Diagnostics 4) Chemstrip Urine Analyzer Roche Diagnostics5) Urisys 1800 System Roche Diagnostics
2) Fullyautomated Chemistry InstrumentsI.Clinitek Atlas Siemens DiagnosticsII.Chemstrip Super Automated Urine Analyser Roche Diagnosticsbl.Urisys 2400 system Roche Diagnostics
3) Automated MicroscopyI.UF-100 Urine Cell Analyser SysmexII.iQ200 Automated Urine Microscopy Iris Diagnostics Division
4) Automated Urinalysis Systems
I.ADVIA Urinalysis Workcell Siemens Diagnostics
II.iQ200 Automated Urinalysis System Iris Diagnostics Division
Clinitek 50/100
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Clinitek 500 • Distinguishes between hemolyzed and nonhemolyzed specimen• Determine low SG and pH• Rapid entry
• Specimen ID• Color• Clarity
• Automatic features• Color determination• Strips detection• Calibration• Confirmatory• Microscopic analysis
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Clinitek Status Analyzer• The Analyzer is for in vitro use in the semi-quantitative detection
of Albumin, bilirubin, blood (occult), creatinine, glucose, ketone (acetoacetic acid), leukocytes, nitrite, pH, protein, specific gravity and urobilinogen in urine samples
• The calculation of albumin-to-creatinine and protein-to-creatinine ratios in urine samples, when Clinitek® Microalbumin and Multistix PRO® Reagent Strips for Urinalysis are used
• The detection of human Chorionic Gonadotropin (hCG) in urine samples, when Clinitest® hCG cassettes are used
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Urine Reagent Strips are made for urinalysis of both qualitative and semi-quantitative, which are in vitro reagent for diagnostics.
The results on the strips can be read visually and instrumentally. The pH and Protein can be read at any
time within 60 seconds after dipping. For a qualitative result, the strip should be read
between 1-2 minutes after dipping .Colour changes beyond 2 minutes are of no diagnostic value.
Reaction Principles
1)Glucose: The glucose oxidized by glucose oxidase catalyzes the formation of glucuronic acid and peroxide hydrogen. Peroxide hydrogen releases oxide (0) under the function of peroxidase. (0) oxidizes Iodide potassium, which makes the colour change.2)Bilirubin: The direct bilirubin and dichlorobenzene diazonium produce azo dyes in a strongly acid medium.3)Ketone: The acetoacetic acid and sodium nitroprusside cause reaction in the alkaline medium, which produces a violet colour.
4) Specific Gravity: Electrolyte (M X) in the form of salt in urine reacts with poly methyl vinyl ether and malefic acid (-COOH), which are weak acid ionic exchangers. The reaction produces hydrogenous ionogen, which reacts with pH indicator that causes the colour change. 5) Blood: Haemoglobin acts as peroxidase. It can cause peroxide release, neo-ecotypes oxide (O) oxidizes the indicator and makes the colour change subsequently.
6) pH: The method of the pH indicator is applied.7) Protein: This is based on the protein-error-of-indicator principle. Anion in the specific pH indicator attracted by caution on the protein molecule makes the indicator further Ionized, which changes its colour.8) Urobilinogen: Urobilinogen and diazonium produce pink azo dyes under the function of a strong acid medium. 9) Nitrite: Nitrite in the urine and aromatic amino sulphanilamide are diazotized to form a diazonium compound. The diazonium compound
reacting with tetrahydro benzo (h) quinolinphenol causes the colour change.10 )Leukocytes: Granulocyte leukocytes in urine contain esterase’s that catalyze the hydrolysis of the pyrrole amino acid ester to liberate 3-hydroxy 5-pheny pyrrole. This pyrrole reacting with diazonium forms a purple colour.
Reactive ingredients (based on dry weight at time of impregnation)oProtein: 0.1% w/w tetrabromphenol blue; 97.4% w/w buffer: 2.5/) w/w non reactive ingredients.oBlood: 26.0% w/w diisopropylbenzene dihydro peroxide; 1.5% w/w tetramethylbenzidine; 35.3% w/w buffer; 37.2 % w/w non reactive ingredients.oGlucose: 1.7% w/w glucose oxidase (microbial 123U); 0.2% w/w peroxidase (horseradish. 203 IU); 0.1 % w/w potassium iodide; 71.8% w/w buffer; 26.2% w/w non reactive ingredients.oKetone: 5.7% w/w sodium nitroprusside; 29.9% w/w non reactive ingredients: 64.4% w/w buffer.oLeukocytes: 4.3% w/w pyrrole amino acid: 0.4% w/w diazonium salt; 92.6% w/w buffer, 2.7% w/w non reactive ingredients.
o Nitrite: 1.3% w/w p-arsanilic acid; 0.9% w/w tetrahydroquinoline N-(1-
Naphthol)-ethylenediamine; 89.6%, w/w buffer; 8.2% w/w non
o reactive ingredients.
o Specific Gravity: 4.8% w/w bromthymol blue; 90.2% w/w poly(methyl vinyl
ether co maleic anhydride); 5.0% w/w sodium hydroxide.
o pH: 3.3% w/w bromcresol green; 55.0% w/w bromthymol blue: 41.7% w/w
non reactive ingredients.
o Bilirubin: 0.6% w/w 2.4 -dichlorobenzene amine diazonium salt; 57.3% w/w
buffer; 42.1% w/w non reactive ingredients.
o Urobilinogen: 0.2& w/w fast blue B salt; 98.0% w/w buffer; 1.8% w/w non
reactive ingredients.
Siemens Clinitek Microalbumin 2 Reagent Strips• Provide albumin, creatinine, and albumin to creatinine ratio results
in 1 minute• useful to test for microalbuminuria in patients with
diabetes or hypertension in order to detect early kidney disease
• 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)
Siemens Clinitek Microalbumin 9 Reagent Strips
• Provide albumin, blood, creatinine, 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
Dirui H-500 Urine Analyzer
The Dirui H-500 Urine Analyzer provides results for urine
testsamples based on an advanced high luminosity cold
light source with 4-wavelength technology. This improves
sensitivity, accuracy and specificity and reduces ambient
light interference.
Automatic waste handling avoids sample cross-
contamination and the analyzer offers a quiet, high speed
built-in thermal printer or external stylus printer
Additional Information about the H-500
Test wavelength: 525nm, 572nm, 610nm, 660nmOffers a test throughput of 514 strips per hourProvides a data memory of 5,000 patient resultsA 5.7″ LCD display provides ease of useComes with a built-in high speed, low noise thermal printerContinuous feedFlag abnormal values
Test ItemsUrobilinogen, Bilirubin, Ketone, Blood, Protein, Nitrite, Leukocytes, Glucose, Specific gravity, pH
+ Urobilinogen+ Bilirubin+ Ketone+Blood+Protein - Nitrite
++ Leucocytes
+++ Glucose1.020 SG
7.0 pH
Used Strip Fresh Strip
URISYS 2400 system Chemstrip super automated
• Technique
Reflectance Photometry - uses the principle that light
reflection from the test pads decreases in proportion to the
intensity of color produced by the concentration of the test
substance.
A monochromatic light source is directed toward the
reagent pads.
The light is reflected to a photodetector and an
analog /digital converter.
The ultimate goal of automation is to improve
reproducibility and color discrimination, increasing
productivity and standardization for reporting
urinalysis results
The Chemstrip Super Automated Urine Analyser and
the Roche Diagnostics Urisys 2400 system are fully
automated ‘walk-away’ urine chemistry instruments for
a large urinalysis laboratory.
With the Chemstrip Super Automated Urine Analyser,
sample volumes are detected, adjusted and
automatically mixed.
• A sorter mechanism supplies a single test strip from
the sorter drum to a sorter position.
• A gripping mechanism grasps the test strip and dips
it in to the urine specimen tube.
• The dipping mechanism lifts the test strip out of
the sample tube while removing excess urine by
dragging the strip along the inside of the
specimen tube
• The dipping mechanism then transfers the test
strip to the Reflectance Photometer position.
• A transport plate positions the test strip at the
Reflectance Photometer recording head, where specimen
is measured at three different wavelengths (555,620,660
nm) at 48 seconds and 120 seconds after dipping.
• The Urisys 2400 system utilizes a pippetting unit that
automatically mixes the specimen and pipettes the
precise volume to each test pad.
• The minimum sample volume is 1.5 ml. Four hundred test
strips are loaded into a Urisys 2400 cassette and the strips
are stable in the cassette for 2 weeks. 75 samples per load
for immediate measurement of emergency samples.
• It designed for large Lab
• Performs >12 tests automatically
• Walk-away capability (> 225 specimen/hr)
• > 2 mL urine specimen required
• Flagging abnormal specimen
• Automatic features• Color determination• Strips detection• Calibration• Confirmatory• Microscopic analysis• etc
Clinitek AtlasClinitek Atlas
Automated MicroscopyAutomated Urine cell analyzers provide efficient
standardized results in about a minute, markedly improving
turnaround times.
The Sysmex UF – Series offers fully automated sample
analysis with automatic classification of all 10 formed
element groups with scattergrams and histograms for
reference.
Disadvantages of Manual Urine Sediment Microscopy
o Subjective element identificationo Poor reproducibilityo Lack of standardizationo Time consuming/labor intensive
• The UF-100 and UF –50 use laser – based flow
cytometry along with impedance detection, forward
light scatter and fluorescence to identify the individual
characteristics and stained urine sediment particles in
a flowing stream.
Sysmex UF 50 Sysmex UF 100
The sample is stained with two dyes that radiate an
orange and green fluorescence. The DNA within the cells
is stained by the orange dye, phenathridine ; the nuclear
membranes, mitochondria and negatively charged cell
membranes are stained with a green dye, carbocyanine
The stained sample is passed through the flow cell,
presented to a laser light beam that produces
fluorescence and light scatter
• The main parameters are RBCs, WBCs, epithelial cells, casts
and bacteria. Flagging parameters include pathologic casts,
crystals, small round cells, sperm and yeast like cells.
• Particles are identified by measuring the change in
impedance of the sediment elements, as well as the
height and width of the fluorescent and light scatter
signals, presented in scattergrams and histograms.
IQ 200 Automated Urine microscopy analyzer
Automatically analyzes and Classifies urine particles
in to 12 categories. The sample is mixed and
aspirated to a planar 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 casts, unclassified casts,
squamous and non Squamous epithelial cells, bacteria,
yeast, crystals, mucus and sperm
IRIS Flow Videomicroscopy • Urine is drawn through a flat
chamber• Video snaps are sorted by
computer• Technician scans images and
deletes dud onesComputer then adds up #/cmm
• These are RBCs52
IRIS Flow Videomicroscopy
• Squamous epithelial cells
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Accuracy of the iQ200 and UF-100 systems in comparison with microscopic results.
Parameter Accuracy (95% CI), % iQ200 UF-100
Leukocytes 89 (85.5–92.5) 84 (80–88)Erythrocytes 86 (82–90) 81 (77–85)Bacteria 68 (63–73) 42 (36.5–47.5)Pathological casts 91 (88–94) 86 (82–90)Yeasts 97 (95–99) 93 (90–96)Crystals 92 (89–95) 88 (84–92)
Sensitivity and specificity of the systems calculated based on the cut-off values.
iQ200 Sensitivity % Specifity % NPV % PPV %
Leukocytes 76 97.5 94 89Erythrocytes 70 98 90 92Bacteria 85 95 88 94Pathological 68 97 93 83castsYeasts 70 99 97 91Crystals 71 97 95 80
UF-100Leukocytes 92 90 98 65Erythrocytes 76 93 92 78Bacteria 95 60 96 53Pathological 57 96 90 78castsYeasts 62 97 96 66Crystals 51 99 88 95
Automated Urinalysis Systems
• Clinitek Atlas, an automated urine chemistry analyzer
and the Sysmex UF-100, an automated urine cell
analyzer, have been integrated to develop the ADVIA
Urinalysis Workcell System.
Sysmex UF-1000i
Sysmex UF-1000i
• Laser-based flow cytometer utilizing 2 stains with fluorescent dyes to stain cellular elements
• Separate bacteria channel for improved discrimination• Forward scatter, hydrodynamic focusing, forward
fluorescent light, conductivity measurements, and adaptive cluster analysis
Sysmex UF-1000i System Components
• 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
The Sysmex® UF-1000i, an automated urine particle analyzer, is a dedicated system for the analysis of microscopic formed elements in urine specimens. The instrument consists for three principal units: (1)Main Unit which aspirates, dilutes, mixes and analyzes urine samples;(2) Auto Sampler Unit supplies samples to the Main Unit automatically;
(3) IPU (Information Processing Unit) which processes data from the Main Unit and provides the operator interface with the system. The UF-1000i is equipped with a Sampler that provides continuous automated sampling for up to 50 tubes.
• The instrument utilizes Sysmex flow cytometry using a red semiconductor laser for analyzing organized elements of urine.
• Particle characterization and identification is based on detection of forward scatter, fluorescence and adaptive cluster analysis.
• Using its own reagents, the UF-I000i automatically classifies organized elements of urine and carries out all processes automatically from aspiration of the sample to outputting the results.
UF-1000i Tube Rotator
UF-1000i Reagents
UFII SEARCH™-SED
UFII PACK™-SEDUFII SHEATH™
UFII SEARCH™ -BAC
UFII PACK™-BAC
UF II PACK-SED / UF II SEARCH-SED
UF II PACK-SEDRemoval of amorphous salts together with heating (up to 35°C)
UF II SEARCH-SEDPolymethine dyeChromogen chain with electron donor and acceptor group
Stains parts of nucleus, parts of cytoplasm and membranesExcitation wavelength is 635 nmEmission 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
Flow cell
Laser light
Laminar Flow
particlesSheath reagent
Sheath nozzle
Scattered light
UF-1000i Detection Parameters
MucusBacteria
Sperm
YeastHyaline Casts
Small Round CellsEpithelial Cells
CrystalsWBC
Pathological CastsRBC
Flagged ParametersEnumerated Parameters
RBC
Small - medium size
Low fluorescenceFl
Fsc
Fl
FscMedium - large size
Medium - high fluorescence
Fl
FscVery small size
Siz
e (s
ectio
nal a
rea)
Large
Small
Fluorescence HighLow
Bacteria
WBC
Low fluorescence
S_FLH
S_FscS1: FLH / Fsc - Scattergram
YLC
X’TAL
Sperm
Low to medium fluorescenceFl
FscSmall size
Medium fluorescenceFl
FscSmall size
Fl
FscSmall - large size
no fluorescence
YLC
RBC
Small - medium size
Low fluorescenceFl
Fsc
Fl
Fsc
Fl
Fsc
Siz
e (s
ectio
nal a
rea)
Large
Small
Fluorescence HighLow
Bacteria
WBC
Low fluorescence
S_FLL
S_Fsc
S2: FLL / Fsc - Scattergram
Low to medium fluorescenceFl
Fsc Small size
Medium fluorescenceFl
FscSmall size
Fl
FscMedium – very large size
Medium - high fluorescence EC
SpermVery small size
Medium - high fluorescence
Medium - large size
S3: FLLW2 / FLLW - Scattergram
FLLW2
S_FLLW2Large
Small
Length of stained particleShort S_FLLW
FLLW
FLLW2
Little to more stainable inclusions
FLLW
FLLW
Long
Length of stained inclusions
Casts (no inclusions)Leng
th o
f sta
ined
incl
u sio
ns
Short – medium lengthof inclusions No to little inclusions
More stainable inclusions
Path. casts
Epithelial ce
lls
Mucus
FLLW2
FLLW
No inclusions
SRC
WBC
FLLW2
Long
B1: Fsc / FLH - ScattergramB_FSC
Large
Small
Stainability of particlesLow B_FLH
Siz
e of
par
ticle
s
BACTDebris
Weak fluorescenceFlH
FscSmall size
FSC
FLH
Small to big size
No fluorescence
High
UF-1000i Sediment 1
UF-1000iSediment 2
UF-1000iSediment 3
UF-1000iBacteria 1
UF-1000iBacteria 2
UF-1000iBacteria 3
UF-1000i Technology
BacteriaSediments
BacteriaSediments Stain
IncubationIncubation
DetectionDetection unitunit
Sediments Bacteria Diluents
Two chambers for stain and dilution
Improved determination of bacteria
Red semiconductor laser•Down sizing•Long life•Reduced power consumption
Sample Incubation
• Incubation time at certain temperature ranges needed for staining – for the SED analysis:
• 10 seconds at 35°C– for the BAC analysis:
• 20 seconds at 42°C
UF-1000i Technology
Fluorescence
Stain DNA/RNA
Non-specific staining with debris
Forw
ard
Scat
ter Specific stain for Nucleic Acid Specific stain for Nucleic Acid
DyeDye
Dye
DyeDyeDye
Dye
DyeDyeDyeDye
Dye
Dye
Dye
DyeDye
Dye
Dye
Dye
DyeDyeDye
DyeDye
DyeDyeDyeDye
1) Enhanced detection of bacteria
2) Staining bacteria nuclei
Polymethine dye
UF-1000i Technology
UF-100 :RBC 119.8/µLX’TAL 0.0 /µL
UF-1000i: RBC 3.3/µL X’TAL 102.7/µL
Microscopy :RBC 5.6/µLX’TAL (2+)
false-positive by X’TAL interferenceReduction of false-positive by X’TAL interference to RBCScattergram
UF-100UF-1000i
The more complex the surface or inner construction, the more intensive SSC signal is.
S-FSC
UF-1000i Technology
UF-100 :EC 83.4/µL
UF-1000i: EC 18.2/µL
Microscopy:EC 24.5/µL
WBC cluster can be detected as EC. It is false positive of EC.
Reduced false positive EC with high positive WBC
Scattergram
UF-100UF-1000i
SSC parameters can help UF to distinguish WBC and EC.
WBC is accurately classified by SSC signals.
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