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

8

9

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

15

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

16

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

17

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

53

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.

ReferencesBerger D. A brief history of medical diagnosis and the birth of the

clinical laboratory Part 1—Ancient times through the 19th century [Internet]. 1999. p. 1–8. Available from: http://www.academia.dk/Blog/wp-content/uploads/KlinLab-Hist/LabHistory1.pdf

Karcher DS, McPherson RA, Pincus MR. Urinalysis. In: McPherson RA, Pincus MR, eds. Henry’s Clinical Diagnosis and Management by Laboratory Methods. 23rd Ed. China: Elsevier; 2017. p. 441–80.

Richard Thompson, Andrew Gammie, Debbie Lewis, Rebecca Smith CE. Evidence review: Automated urine screening systems. CEP10030. Cent Evidence-based Purch. 2010;1-46.

Shayanfar N, Tobler U, Von Eckardstein A, Bestmann L. 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 [Internet]. 2007;45:1251–6. Available from: http://doi.org/10.5167/uzh-81721

References• Strasinger SK, Lorenzo MS Di. Automated Urinalysis. In: Strasinger

SK, Lorenzo MS Di, ed. Urinalysis and body fluids. 5th Ed. Philadelphia: F.A. Davis; 2008. p. 259–63.

• Chien TI, Kao JT, Liu HL, Lin PC, Hong JS, Hsieh HP, et al. Urine sediment examination: A comparison of automated urinalysis systems and manual microscopy. Clin Chim Acta. 2007;384:28–34.

• Ben-Ezra J, Bork L, Mcpherson RA. Evaluation of the Sysmex UF-100 automated urinalysis analyzer. Clin Chem. 1998;44:92–5.

• Budak YU, Huysal K. Comparison of three automated systems for urine chemistry and sediment analysis in routine laboratory practice. Clin Lab [Internet]. 2017;57:47–52. Available from: http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=21391464