2 - AU Assay Introduction

AU ASSAY INTRODUCTION
1 © 2017 Beckman Coulter. All rights reserved.

© 2017 Beckman Coulter. All rights reserved.
MANUFACTURER SITE OF CC PRODUCTS

MISHIMA, JAPAN

CLARE FACILITY, IRELAND

AU REAGENTS MANUFACTURE IN CLARE
•Reagent •QC •Calibrator •Cleaning solution

AU REAGENT
• OSR (Olympus System

Reagents ) 120ml
• Different packing match to
with customer workload 60ml
180ml
 15ml bottle
 30ml bottle
 60ml bottle
 120ml bottle
 180ml bottle 30ml
15ml
AU REAGENT BOTTLE
Adapter
• 15 mL and 30 mL bottle
adapters
Partition
Adapter
• Partitions, can be manually

adjusted to adopt different
size of reagent

AU REAGENT BOTTLE

BARCODE SCANNING
• Reagent Name
• Test quantity
• Onboard exp date
• Calibration exp date
• Reagent expiry date
• Lot/ serial number

BENEFIT OF CONCENTRATED REAGENT
• Reduce frequency of reagent loading
• Increase workflow efficiency
• Maximize onboard reagent capacity
• Reduce reagent usage
• No preparation needed, analyzer will perform auto
dilution

AGENDA
1. Photometry
• End point reactions
• Kinetic reactions
• Immunoturbidimetry
• EMIT
• Latex agglutination
2. Ion selective electrodes
3. Chemistry Reagent - Same test with different methods

AU REACTION THEORY - COLOURIMETRY
• Colourimetric assays are designed to detect or quantitate the amount of a
particular analyte in a sample by measuring the amount of light absorbed by the
analyte (or more commonly its coloured reaction product) at a characteristic
wavelength.
• This wavelength is specific to the analyte being measured. Typically, the greater
the amount of analyte present, the more light will be absorbed. Many colorimetric
assays utilize a chromogenic (colour-producing) substrate that, when converted
into its final product (called a chromophore), will absorb light at a specific
wavelength.
• The AU detects 13 wavelengths from 340nm to 800nm.

AU REACTION THEORY - PHOTOMETRY
Spectrophotometry is a method to measure how much a chemical substance

absorbs light by measuring the intensity of light at particular wavelengths after a
beam of white light passes through sample solution. Colourimetric assays are run
on a spectrophotometer. The AU system is therefore a high throughput
spectrophotometer.
Components involved:
› Lamp
› Diffraction grating
› Photodiode array

Lamp:
› Provides the white light source
Diffraction grating:
› A diffraction grating is an optical component that splits
light into several beams/wavelengths
Photodiode array:
› A photodiode is a semiconductor device that converts light into current
› The AU photodiode array has diodes specific for 13 wavelengths from 340nm to 800nm
› These are the wavelengths used by the assays.

AU5800 AU480/680/DxC700AU

There are 28 read points taken at 18 second intervals during the 8.5
minutes of each reaction. This diagram shows the reaction process -
what is happening in a each cuvette.
Each assay uses different read points to determine concentration.
• R1 Dispensing
• R1 Mixing
• P0 (first cuvette OD read point, R1 reagent only)
• Sample Dispensing
• Sample Mixing
• P1 (OD read point sample + R1 reagent)
• P2-P9 (OD read point intervals, 18 seconds)
• P10 (last OD read of R1 + sample before the addition of R2)
• R2 Dispensing
• R2 Mixing
• P11 (first cuvette read point with R1 + sample + R2)
• P12-P26
• P27 (final read point)

TYPE OF ASSAY METHODS
Methodology Assay Example
End Point Assay Calcium Arsenazo,
(One point assay) Albumin
End Point Assay Glucose, Phosphate,

(Two point assay)
Rate Assay Most enzymatic assays –

ALP, AST ALT, LDH etc
Fixed Point Bicarbonate and

Assay creatinine

ASSAY METHODS– END POINT ASSAYS
1-Point Assay
› Single Step Assay
› Difference between the initial OD and a specific point OD
› Examples: mostly R1 assays, Calcium, Albumin

2-Point Assay (self-blank method)

› Sample blank adjustment
› Corrects for OD change before R2 addition
› Examples: Glucose, Phos

TYPES OF ENDPOINT ASSAYS
Slow Endpoint Fast Endpoint
Triglyceride,Cholesterol,Glucose etc Iron, Albumin,TP, etc

› A reaction takes place and ends after a set time (it reaches the endpoint!).
› The “Last” Endpoint measuring point is picked under robust conditions, i.e. at
the end of the shelf-life of reagent
› Slow end-point reactions usually have P27 as last measuring point, why have it
any earlier!
› For fast endpoint reactions, the reaction is usually completed before P27
› During development active ingredients /substrate concentrations/activator levels
would be adjusted to make reactions happen faster/slower if this was required.
End Assay (Sample blank correction)

› Sample blank adjustment
› Corrects for sample quality issues
› Uses 2 cuvettes: colour reaction + sample blank
› Uses Examples: Bilirubin assays

ASSAY METHODS – RATE ASSAYS
Rate assay
› Rate of absorbance variation per minute
› Variation between two points calculated by least square method
› Some rate assays can be calibrated using a fixed calibration factor.
› Examples: Most enzymatic assays – ALP, AST ALT, LDH etc
› The enzyme unit (U) is a unit for the amount of a particular enzyme.
One U is defined as the amount of the enzyme that produces a certain
amount of enzymatic activity, that is, the amount that catalyzes the
conversion of 1 micro mole of substrate per minute.

ASSAY METHODS– FIXED POINT ASSAY
Fixed Point Assay

› Measures OD between two
specified points
› Both points are measured after
reaction has begun
› Examples: Bicarbonate and
creatinine

EMIT
 Enzyme multiplied immunoassay technique(EMIT) is a common

method for qualitative and quantitative determination of drugs
 Fast
 Reliable
 Accurate
 Applications for both AU 8 Series and DxC700 AU
• Gold Standard
• Suitable for all testing volumes
• Legal defensibility

PRODUCT OFFERING
Beckman/Siemens Co-Branded Products Siemens Branded off-the-shelf Products

 26 TDM/DAT Co-Branded reagents  11 Reagents, 7 controls and 33 calibrators
 Packaged in AU bottles, have Beckman branding  Siemens off-the-shelf kits NOT packaged in AU
and state Siemens as the legal manufacturer bottles
 Products are barcoded and part of the closed  These products must be configured in open
system concept channels (in a reagent fixed position)
 Registered by Siemens in certain areas

PRODUCT OFFERING EXCEPTIONS
• OSR6404 Digoxin from Thermo CEDIA will continue to be offered to ALL OUS
customers on both AU 8 Series & DxC 700 AU
• OSR4H229 Digoxin from Siemens Syva will be only offered to US customers on
Digoxin both AU 8 Series & DxC 700 AU
• Siemens does not offer Digitoxin reagent, the request should be fulfilled by
Thermo/Clare OSR6403 Digitoxin
• OSR6318 EDDP products will continue to be sold and offered by Thermo/Clare
Digitoxin &
EDDP • Major market is Germany
• OSR61202 Acetaminophen from Cambridge for OUS will be obsoleted and

converted to Siemens Syva Acetaminophen OSR7A229 globally
Acetaminophen • Transition is being planned and will be implemented soon
 Lithium and Ammonia are not part of the Siemens roll-out

 Will continue to offer Thermo products

COMPREHENSIVE TDMS/DATS MENU OFFERING

EMIT REACTIONS FOR TDM AND DATS
 Quantitative analysis of the drug in human serum or plasma

 The assay is based on competition between drug in the sample and drug
labeled with the enzyme glucose-6-phosphate dehydrogenase (G6PDH) for
antibody binding sites.
 Enzyme activity decreases upon binding to the antibody, so the drug
concentration in the sample can be measured in terms of enzyme activity.
 Active enzyme converts oxidized nicotinamide adenine dinucleotide (NAD) to
NADH, resulting in an absorbance change that is measured
spectrophotometrically at 340nm
Coenzyme
Enzyme-labeled
Antibody specific analyte
to analyte
Coenzyme conversion
Analyte Converted
producing absorbance coenzyme
change
Enzyme substrate
DRUG-G6PDH
(active)
Glucose-6-phosphate + NAD+ 6-Phosphogluconolactone + NADH + H+
(absorbs at 340 nm)
CEDIA REACTION
Cloned enzyme donor immunoassays
› Quantitative analysis of the drug in human serum or plasma
› The assay is based on competition between drug in the sample and
drug labeled with the enzyme glucose-6-phosphate dehydrogenase
(G6PDH) for antibody binding sites.
› Enzyme activity decreases upon binding to the antibody, so the drug
concentration in the sample can be measured in terms of enzyme
activity.
› Active enzyme converts oxidized nicotinamide adenine dinucleotide
(NAD) to NADH, resulting in an absorbance change that is measured
spectrophotometrically at 340nm

CEDIA REACTION
No ligand/analyte present in sample
Ligand/Analyte present in sample

IMMUNOTURBIDIMETRY
Example:
Syphilis and
Procalcitonin

ION SELECTIVE ELECTRODES
• Analyte concentration is
calculated by measuring
the potential difference
across a membrane via the
Nernst Equation
• ISE’s are used by all
manufacturers as a fast,
accurate and precise way
of measuring electrolytes
• Generally require special
maintenance and frequent
calibration

CHEMISTRY
REAGENTS

SAME TEST – DIFFERENT METHOD
There are some assays where it is possible to sell reagents with differing
assay principles
›Creatinine Jaffe V Enzymatic

›AST/ALT With or without Pyridoxyl phosphate
›LD Pyruvate to Lactate V Lactate to Pyruvate
›Calcium Arsenazo V CPC

ALT/AST
• The transaminases
• These enzymes catalyse the transfer of amino acids
• Pyridoxyl Phosphate (P5P) acts as a co-enzyme in the amino transfer
reactions
• P5P is vitamin B6
• The reagent does not contain P5P because almost everybody has
vitamin B6.
• If a patient is Vitamin B6 deplete, which occurs with alcoholism will
lead to a decrease in the activity of the transaminases
• This will also extend to the testing method
• To avoid this, we can add P5P to the reagent.
• This will avoid falsely low ALT and AST results

AU680/5800/DxC700AU
AU Processchart
Process chart
R1
R1-1 R2
R1-2 Sample
MIX MIX MIX
M.P 0 1 ........ 10 11 12 ........ 27
Why is three-step testing necessary ?

Three-Step Reagent Testing
IFCC RECOMMENDATIONS FOR ALT AND AST
• The coenzyme Pyridoxal 5 Phosphate (vitamin B6) is required to be

added while testing ALT and AST
• But mixing P5P with R1 reagent will hasten the deterioration of the
reagent.
• By three-step testing method, P5P will be dispensed separately during

the main reaction which will not affect the on-board stability of the
reagent.

Three-Step Reagent Testing
AST (IFCC, WITH OSR60180 ACTIVATION ), AU680

LD
• IFCC recommended method OSR6X28

Lactate + NAD+ is catalysed by LD to form pyruvate + NADH + H+
OR
• Reverse reaction OSR6X26

Pyruvate + NADH + H+ is catalysed by LD to form Lactate + NAD+
• The IFCC method will yield LD activities which are approximately 65

% of those form the alternate method

MEASUREMENT OF CREATININE
• The Jaffe reaction

• Jaffe first described this method in 1886 when it was published in Z
Physiol Chem 1886;10:391-400
Creatinine + Picric acid Alkaline pH yellow complex
• This will measure any compound that reacts with picrate in alkaline
conditions
• Some reacting compounds: protein, albumin, ketones, bilirubin,
cephalosporins, ...

ACCURACY IN CREATININE MEASUREMENT
Greenwald J, McGuire G. The estimation of creatine and creatinine in the blood J

Biol Chem 1918;34:103
› “For an investigation of normal creatine and creatinine metabolism, the methods
are probably not satisfactory. Until it can be shown that the chromogenic substance
is really creatinine, investigations in this field would seem to be of doubtful value”
Behre JA, Benedict SR. Studies in creatine and creatinine metabolism IV. On the
question of the occurrence of creatinine and creatine in blood. J Biol Chem
1922;52:11-33
› “ A review of the literature on methods of creatinine determination in blood cannot
but leave one with the impression that each investigator, using a new technique
and one seemingly accurate, is able to get figures quite different from those
obtained by any previous method”

ENZYMATIC METHODS
Creatinine + H2O creatininase Creatine
Creatine + H2O creatinase Sarcosine + Urea
Sarcosine + O2 + H2O sarcosine oxidase Formaldehyde + Glycine + H2O2
The H2O2 generated can be then measured using a Trinder’s reaction.

CREATININE
• Enzymatic creatinine is more accurate than Jaffe

• There are less interferences with Enzymatic Creatinine
• Enzymatic Creatinine is > 5 times the price of the Jaffe
• More labs are changing to the enzymatic assay but the cost is hard to
justify
• If a customer is very technical it is possible to convince them to look at
the enzymatic method

CREATININE INTERFERENCES AND LIMITATIONS
Interferent Concentration Bias Jaffe rate blanked method Bias Enzymatic Method
Ascorbate 10 mmol/L + 37 umol/L - 13 umol/L
Pyruvate 2 mmol/L + 31 umol/L Not significant
Glucose 50 mmol/L + 19 umol/L Not significant
Creatine 2 mmol/L + 16 umol/L Not significant
HbF 10 g/L - 66 umol/L Not significant
Dopamine 1 mmol/L -15 umol/L - 66 umol/L
Cephaolsporins 1 mmol/L 0 – 213 umol/L Not significant
Table from Peake M, Whiting M; Clin Biochem Rev ;Vol 27 November 2006
CALCIUM CPC VS ARSENAZO III

THANK YOU!

Beckman Coulter, the stylized logo and the Beckman Coulter product and service
names mentioned herein are trademarks or registered trademarks of Beckman Coulter,
© 2017
48 in the
Inc. Beckman
United StatesCoulter.
and All rights
other reserved.
countries.
©2017 Beckman Coulter. All right reserved. Beckman Coulter Confidential.

2 - AU Assay Introduction

Uploaded by

Document Information

Original Title

Copyright

Available Formats

Share this document

Share or Embed Document

Sharing Options

Did you find this document useful?

Is this content inappropriate?

Copyright:

Available Formats

2 - AU Assay Introduction

Uploaded by

Copyright:

Available Formats

AU ASSAY INTRODUCTION

1 © 2017 Beckman Coulter. All rights reserved.

2 © 2017 Beckman Coulter. All rights reserved.

3 © 2017 Beckman Coulter. All rights reserved.

4 © 2017 Beckman Coulter. All rights reserved.

•Reagent •QC •Calibrator •Cleaning solution

5 © 2017 Beckman Coulter. All rights reserved.

• OSR (Olympus System

• Partitions, can be manually

7 © 2017 Beckman Coulter. All rights reserved.

8 © 2017 Beckman Coulter. All rights reserved.

9 © 2017 Beckman Coulter. All rights reserved.

12 © 2017 Beckman Coulter. All rights reserved.

2. Ion selective electrodes

3. Chemistry Reagent - Same test with different methods

13 © 2017 Beckman Coulter. All rights reserved.

• The AU detects 13 wavelengths from 340nm to 800nm.

14 © 2017 Beckman Coulter. All rights reserved.

Spectrophotometry is a method to measure how much a chemical substance

15 © 2017 Beckman Coulter. All rights reserved.

16 © 2017 Beckman Coulter. All rights reserved.

17 © 2017 Beckman Coulter. All rights reserved.

Each assay uses different read points to determine concentration.

18 © 2017 Beckman Coulter. All rights reserved.

End Point Assay Glucose, Phosphate,

Rate Assay Most enzymatic assays –

Fixed Point Bicarbonate and

19 © 2017 Beckman Coulter. All rights reserved.

20 © 2017 Beckman Coulter. All rights reserved.

2-Point Assay (self-blank method)

21 © 2017 Beckman Coulter. All rights reserved.

Triglyceride,Cholesterol,Glucose etc Iron, Albumin,TP, etc

End Assay (Sample blank correction)

23 © 2017 Beckman Coulter. All rights reserved.

24 © 2017 Beckman Coulter. All rights reserved.

Fixed Point Assay

25 © 2017 Beckman Coulter. All rights reserved.

 Enzyme multiplied immunoassay technique(EMIT) is a common

26 © 2017 Beckman Coulter. All rights reserved.

Beckman/Siemens Co-Branded Products Siemens Branded off-the-shelf Products

27 © 2017 Beckman Coulter. All rights reserved.

• OSR61202 Acetaminophen from Cambridge for OUS will be obsoleted and

 Lithium and Ammonia are not part of the Siemens roll-out

28 © 2017 Beckman Coulter. All rights reserved.

29 © 2017 Beckman Coulter. All rights reserved.

 Quantitative analysis of the drug in human serum or plasma

31 © 2017 Beckman Coulter. All rights reserved.

No ligand/analyte present in sample

Ligand/Analyte present in sample

32 © 2017 Beckman Coulter. All rights reserved.

33 © 2017 Beckman Coulter. All rights reserved.

34 © 2017 Beckman Coulter. All rights reserved.

35 © 2017 Beckman Coulter. All rights reserved.

›Creatinine Jaffe V Enzymatic

36 © 2017 Beckman Coulter. All rights reserved.

37 © 2017 Beckman Coulter. All rights reserved.

M.P 0 1 ........ 10 11 12 ........ 27

Why is three-step testing necessary ?

IFCC RECOMMENDATIONS FOR ALT AND AST

• The coenzyme Pyridoxal 5 Phosphate (vitamin B6) is required to be

• By three-step testing method, P5P will be dispensed separately during

39 © 2017 Beckman Coulter. All rights reserved.

AST (IFCC, WITH OSR60180 ACTIVATION ), AU680