CLIA.pdf

Chemiluminescence
immunoassay analyzer
by sohail khan
1

Learning Objectives
✘ Principles
✘ Operation
✘ Sample requirement
✘ Maintenance
✘ calibration
2

Chemiluminescence immunoassay analyzer
✘ Immunoassay – Tests based on specific binding that occurs
between an antibody and antigen that it specifically recognizes
✘ Multiplemethods are available
3

✘ Predominant
 ELISA
4

✘ Used to estimate analytes which have extremely low concentration
in the blood such as
 Hormones
 Serological markers
 drugs
5
chemiluminescence Immunoassays

✘ Is a method to determine the concentration of
samples according to the intensity of the
luminescence that a chemical reaction emits
6

✘ The followingreaction sequence illustrates
the basic principles of a reaction ;
Sandwich immunnoassay by CLIA
7

Sandwich immunoassays by CLIA
( Pateint/ controlsample)
✘ Microparticle reagent is mixed with the patient sample (Ag in this case)
8

Antigen Immobilized Antibody
( Patient/ controlsample) (Reagent)
✘ The micro particle are paramagnetic and coated with capture molecules
(Immobilized Antibody, reagent)
9

immune complex
✘ The reaction mixture incubates and analytes present in the sample binds
to the corresponding capture molecules on the micro particle forming the
immune complex
10

Magnet attracts the paramagnetic particles bound to the specific analytes
To
The wall of the reaction vessel
(washing at this stage removesall unbound materials )
11

conjugate immune complex
(Reagent)
✘ A chemiluminescent acridinium – labelled conjugate is now addedwhich
binds to the immune complex to complete The reaction mixture.
12

sandwich
✘ A chemiluminescent acridinium – labelled conjugate is now addedwhich
binds to the immune complex to complete The reaction mixture.
13

After period of incubation
Reaction mixture is again washed to remove unbound materials
14

sandwich
✘ Pre trigger is added next
15

acidic medium
sandwich
✘ Pre- trigger creates an acidic environment to prevent early release of energy or light
emission. 16

+
substrate
✘ Trigger solution now addedto complete the reaction.
17

Chemiluminiscent reaction
✘ The resulting chemiluminscent reaction is measured as relative light unit
(RLU) .
18

✘ A direct relationship exists between the amount of Ag or Ab in the sample.
19

✘ RLU – Detectedby Architect system optics
20

Signal
concentration of
antigen/Analytes
✘ The generated signal (in RLUs) is directly proportional to conentration of
analyte and computedaccordingly.
21

✘ Fullyautomated chemiluminscence
immunoassay system
22

23
Fig – 01 imageof CLIATheARCHITECTi1000SR https://www.corelaboratory.abbott/int/en/offerings/brands/architect/architect-i1000SR

✘ Samples can be loaded in the primarysample
tubes
24

✘ The system is capable of detection various analytes such as
 Viral hepatitis markers
 Thyroid gland function markers
 Tumourmarkers
fig- 03 system
26

✘ Example of analytes – TSH (thyroidstimulating hormones)
27

Components
28

✘ Processing centre
 Main activity area of processing
 Samples and reagents are dispensed
Mix into RVs (Reaction vessel)
in the process path where assay
processing is performed.
fig- 04processing centre
29

✘ Probe
 Commonfor reagent and samples .
 Supportedby syringe assembly for
aspiration and dispensing.
Fig -05 probe
30

components
✘ The washing unit which is supported
by a washing pump.
Fig -06washing unit
31

✘ The processing centre is a covered circular track that provides
 IncubationTemp.
fig -07 circular track
32

✘ Disposable reaction vessels (RV)
 Different in different analytical systems
 Some systems have pre- coated RVs
with either the Ags or Abs coated on RV
 If an antigen is to be detected in a sample
then the corresponding Ab will be coated
and vice versa
fig- 08 RV
33

fig-09Reaction vesselloader
34

fig -10 storage
35

components
✘ SSC(system control center)
 Is a computersystem thatprovides
 Software interface to system
 Interface to a host computer
 Review the patient results, control data,
Calibration results, etc fig-11 SSC
36

Fig -12
37

components
✘ Wash buffer storage area
 Provide on board storage of wash
buffer
Fig -13
38

components
✘ Wash buffer storage area
 Provide storage area for
 Solid waste container
 Liquid waste container fig-14wash bufferstoragearea
39

components
✘ Sample handlers
 Is transport system used for
 Calibration
 Patient samples fig-15 samplerhandler
40

Components
 Reagents
 Micro- particle solution
 Conjugate solution
 Diluent
 Pre-trigger
 Trigger solution
41

SampleRequirements
 Precautionsforhandlingspecimen
 Followall usual precautions for collecting blood
by venepuncture.
 Check reagent package for the informationabout
 Specimen collection
 Preparation
 storage
42

SampleRequirements
 Consider
 Clinical specimens contain human – sourced
as potentially infectious
 Reagent
 Calibration
43

Reagent Loading
44

fig-18
45
TSH Reagent Kit Literature for Architect System, Abbott Laboratories.Longford, Abbott Park, Wiesbaden

46
https://youtu.be/V2-mjVkeUW4

Sample Loading
47

Fig-19
48

✘ Sample Loading
 Verify the calibration status of the analytes to be tested.
 Run quality control
 Don’t use the calibrators or control if expiration date is
exceeded
49

✘ Sample Loading
 Sample ID details to be entered manually
in the system.
50

Important – you are responsible for loading the correct
sample in correct position
fig-20
51

Avoid the splashing outside of the sample cups and /
or tubes
fig-21
52

✘ Ensure the number of samples loaded matches the
number of samples in the order
53

Operation
54

fig-22
55

✘ Operation
 Sample and micro particle get thoroughlymixed withthe
help of vortexer and are incubated
56

✘ Operation
 Now the probe aspirants the conjugate and dispenses into
washed RV
 The solution is mixed by the vortexer followed by incubation
and Another wash
57

✘ Operation
 Pre – trigger and trigger is added
 This will trigger the emission of light which is measured in the
Relative light units (RLU)with the help of optics
58

✘ Operation
 RLUs are converted into the ODs by the system
 Reading is given for the analyte
59

Maintenance
60

Fig-23
61

62

63

Maintenance
Daily maintenance in this equipment
(Follow the manufacture’s instruction)
64

Maintenance
65

Fig-24
66

Maintenance
Daily maintenance in this equipment
Vacuum line cleaning – Done by system on command
67

Maintenance
weekly maintenance in this equipment
wash zone probe clean
68

Maintenance
wash cup clean
72

Maintenance
wash zone probe clean
76

Maintenance
monthly maintenance in this equipment
80

Calibration
 Equipment calibrations.
 Done Annually
 Probe
 Optical system verification Calibration as per format
 wash zone aspiration
82

Calibration
 Analyzer is under warranty – Responsibility of manufacturer
 Machine has CM/AMC - Responsibility of agenecy
83

Calibration
 Analytecalibration is done ; when
 Reagent lot changes
 As per calibration stability
 Part of the corrective action in QCfailure
84

Calibration
fig-34 Calibration is in liquid state
85

Calibration
fig- 35 stored at 2-8º c
86

Calibration
 Used dropper to avoid contamination
 Dead volumeis 50 µl
 Depending on the assays calibrated, acid required
calibrator
87

Calibration
✘ For example, if the recommended calibrator volumeis X, then the
total volume of calibrator is required is dead volume + X
88

89 https://youtu.be/V2-mjVkeUW4

Calibration
Afterperforming calibration
RLUs areplottedby eqiupment
90

Calibration
fig- 37 Equipment automatically rejects unacceptableRLUs
91

Calibration
fig- 38 status of thecalibrationis accessed from calibrationstatus screen which clearly
shows whether thecalibrationpassedor failed
92

Calibration
fig- 39 Document thecalibration RLUs
93

Calibration
fig- 40
94

Linearity
 Linearity for the TSH-100
95

Linearity
fig-41 If a sampleexceeds 100- Flag with greaterthansymbol
96

Linearity
 Sample have to be diluted externally as per the laboratory’s
reportable range
 Appropriate calculation are done by taking the dilution
factor in the account
97

Linearity
 Equipment is programmed to perform the dilution
the dilutioninternally
 Sample is runin the dilution mode and the result is read
directly
98

Internal quality control
 Corresponding mean and reference range values are available on
the QC kit insert for particular analyte
 Levy jennings charts are plotted for each QC run
99

external quality control
100

external quality control
 Laboratory should enrol in regular EQAS programme
101

Waste disposal
 Disposed in accordance withBMW disposal regulation
102

✘ References
[1] Automated ChemiluminescenceImmunoassay Analyzer
Yao Yufeng,LuShizhou,HuangBo,Zhao Jianwen
Harbin Instituteof Technology at Weihai,Shandong,264209,China
[2] Architect system en TSH 7K62 49-3481/R3 B7K620‘
[3] Brochure of CLIA i1000sr(https://www.medwrench.com/documents/view/4116/abbott-architect-i1000sr-
brochure)
[4] TSH Reagent Kit Literature for Cobas e601,Roche Diagnostics. Mannheim:2011.
[5] ChemiluminescenceImmunoassay:Basic Mechanism and Applications
Mohammad Anwar-ulAzim, Mizanul Hasan, Israque Hossain Ansari and Faria Nasreen
National Institute of Nuclear Medicine and AlliedSciences,BAEC,BSMMU, Shahbagh, Dhaka
103

thanks!
You can find me at
97sohailkhan@gmail.com
104

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