Transcript OnSite Rapid Test Procedure Training Part 4

OnSite Rapid Test Procedure Training
Part 4
CTK-MK-PPT-R0001 (4)
Disclaimer
This is a general training presentation based on the OnSite Rapid Test
procedure. Trainees should take precautions when performing a specific assay,
and should strictly follow the procedure provided by the test kit.
Training Contents
5
• Product Evaluation
• Additional Information
6
Storage Temperature & Shelf Life
4
• Troubleshooting
Limitation of Rapid Test
• The Assay Procedure and the Interpretation of Assay Result sections
in package insert provided with kits must be followed strictly.
• The test band intensity does not have a linear correlation with the
analyte titer in the specimen.
• A negative result indicates absence of detectable analyte, which
does not preclude the possible of exposure or infection.
• Is a screening test, results should only be interpreted in conjunction
with:
o
Clinical findings
o
Confirmatory test
Limitation of Rapid Test
• Is a qualitative or semi-quantitative device
• Has detection limit
limitation
Example
Troponin I
Detects 0.5 ng/mL tropnoin I
PSA
Detects 4 ng/mL and 10 ng/mL PSA
FOB
Detects 25 ng/ml or 50 ng/mL hHb
• Analyte has genetic variation or strain variation
Variation
Example
Isotype or
serotype variation
Dengue has 4 serotypes. A good test has to be able to detect
all of the serotypes
Genetic variation
Few strains of P.f lack of HRP-II protein, leading to falsenegative results on the P.f HRP-II antigen test
Limitation of Rapid Test
• False Negative
It means a test result indicates no presence of analyte (the result is
negative), when compared to reference test.
Causes
Explanation
Limit of detection (LOD)
Analyte in the specimen is below the LOD
Isotype or serotype
variation
Different Isotype and serotype of analyte in the specimen
can not be detected
Timing to sample
Analyte is not present during very early or very late stage
of disease
Hook effect
Analyte concentration is too high; for example, 10 mg/mL
hemoglobin in fecal specimen masks the binding of antiHb Ab, leads to false negative result
Interference by other Anticoagulants, or medicines present in the sample can
substances
inhibit the reaction, leads to false negative
Limitation of Rapid Test
• False Positive
It means a test result indicates presence of analyte (the result is positive),
when compared to reference test.
Causes
Explanation
Cross reaction
A reaction which occurs when surface antigenic
determinants on different molecules of quite different
sources are identical, so that antibody directed against
one antigen also reacts with another.
Interference
substances
by
other • Some specimens containing unusually high titers of
heterophile antibodies, rheumatoid factor (RF),
HAMA or ANA may affect expected results
• Medicine or chemicals may affect expected results.
For example, in Filariasis, if the patient would have
taken DEC or albendazole, IgG4 level will increase and
it may interfere with the test
Read result exceeds the • Increase of reaction time leads to false positive
defined time window
Troubleshooting
• Internal control line (C line)
Problem
Potential Causes
Action
No control line or
significant decrease
of the intensity of
control line
Damaged device
Retest using a new device
Improper procedure
• Follow procedures in the package insert provided
• Recheck buffer and/or specimen volume
• Read the result at defined time
Device expired or stored
improperly
• Check expiration date of the device
• Do not use beyond expiration date stated
• Check storage temperature records
T and C line antibody are both
from mouse. If the T line is too
strong, the conjugate is used
up by the T line, so C line is
weaker
Repeatedly invalid
results
Defect devices
Use different C line system from the T line, such as
rabbit antibody control line system, while the T line
is mouse antibody system
• Check device for defective packaging
• Verify device quality by external control
• Inform supervisor and CTK
Troubleshooting
• Interpretation of assay result
Problem
Potential Causes
Action
• Use correct specimen type
• Spin specimen and get rid of lipid
• Check for the presence of
interference
• Strictly follow IFU procedure
• Don’t use component from
different kit
Read results exceeded defined in IFU
• Strictly follow IFU procedure
Using distilled water or other inappropriate • Use clinical specimens or provided
solution as external control specimen
control as external control
False negative Wrong specimen type
• Use correct specimen type
Incorrect volume of specimens or buffer
• Strictly follow IFU procedure
Read result less or exceed defined in IFU
• Strictly follow IFU procedure
Specimen is collected too early
• Collect specimen few hours, days
or weeks late
Analyte present below the LOD
• Use more sensitive detection
methods
False positive
Wrong specimen type
Specimen contains unusually high titers of
lipids, RF, human anti-mouse Ab, antinuclear Ab or other inference
Incorrect volume of specimens or buffer
Using components from different kit
IFU: Instruction for Use
Troubleshooting
• External Control
Problem
Potential Causes
External control fails to Wrong procedure to
show accurate results
reconstitute control
Control is not stored as
required
Control is expired
Action
• Strictly follow control IFU
• Store control properly. Return control to
its storage condition immediate after use,
if the control is for multiple use
• Use only valid control
• Storage condition
Problem
Potential Causes
Storage
temperature Storage does not have A/C
exceeds recommended system to control temperature
range
Kit is frozen
External temperature is too
cold during transportation or
accidently keep in freezer
Action
Run external control to verify kit
quality
• Bring up the kit into room
temperature before testing,
• Verify the kit qualify with external
control
Troubleshooting Flow Chart
Unexpected Results
Review Procedure and Repeat
Assay with a New Device
Repeated Unexpected Results
Storage & Test
Environment
Specimen Collection,
Extraction & Storage
Procedure
Review Manufacture Instruction
Limitation
of Test
Test with
Alternative Methods
External Control
Retest with
Different Lot/Device
Report to CTK
Trouble shooting – Follow the Correct Procedure!
• More than 50% of complaints received are caused by incorrect
procedure
o
Potential reasons
 Assay is performed by new, untrained lab technician
 The procedure from other product insert is used
 The insert from old revision is used
 The buffer from a different product is used
 Wrong buffer volume is added
 Wrong specimen transfer device is used
 Wrong read time
o
Suggestions
When a complaint is received from a lab, immediately:
 Go through the procedure point by point with the complainant
 Ask if correct components have been used
Therefore 50% of complaints can be solved instantly!
Complaint Handling Procedure
Customer Complaints
Recorded and Classified the Complaints
by Customer Service & QA
General Complaints
(Not Products Related)
Product Complaints
Evaluated and Processed by
Customer Service
Investigated by QA
Record, Review, Inspection, Reference
Corrective and Preventive
Action if Necessary
Corrective and Preventive
Action if Necessary
Customer Service
Reply to Customer
QA & Product Manager
Reply to Customer
Reviewed and Closed by QA
Check
Procedure
5
• Product Evaluation
Evaluation of Rapid Test
• For sensitivity and Specificity, generally in situation:
Increased desire to detect all truly
positive patients for treatment or
the treatment is not necessary
High Specificity
Increased desire to avoid false
positives if the illness is rare
or treatment is burdensome
to the patient
High Sensitivity
Evaluation of Rapid Test
• Evaluation protocol
Specimen
Collection, Extraction, Storage, Dilution
OnSite Rapid Test
Competitor Rapid Test
Results
Results
Compare
“Apple to Apple”
Evaluation
• Agree
• Disagree: resolve discrepant results
Conclusion
Compare
to 3rd party test
Evaluation of Rapid Test
Performance on BBI Mixed Titer Performance Panel 08454
RDT
OnSite
RDT
competitor
IgM
IgG
Step1
ELISA
Comparison with
competitor RDT
IgM
IgG
Positive
Positive
Positive
Negative
Negative
Negative
Positive
Positive
Positive
Negative
Positive
Step2
Positive
Negative
Positive
Positive
Positive
Positive
Positive
Positive
Positive
Negative
Positive
0051-11
Negative
Positive
Negative
Negative
Negative
Positive
Resolve difference
By ELISA
0051-12
Negative
Positive
Negative
Negative
Negative
Positive
0051-13
Negative
Positive
Negative
Positive
Negative
Positive
Conclusion:
0051-14
Negative
Negative
Negative
Negative
Negative
Negative
0051-15
Negative
Positive
Negative
Negative
Positive
0051-16
Positive
Positive
Positive
Positive
Positive
Negative
Positive
0051-17
Positive
Positive
Positive
Positive
Positive
Positive
0051-18
Negative
Positive
Negative
Negative
Negative
Positive
0051-19
Negative
Positive
Negative
Negative
Negative
Positive
• OnSite RDT is more accurate than
competitor’s test, by using ELISA as
confirmatory test
• It could mislead if ELISA test was
not used to verify the discrepancy
specimens
0051-20
Positive
Positive
Positive
Positive
Positive
Positive
0051-21
Positive
Positive
Positive
Positive
Positive
Positive
IgM
IgG
0051-01
Positive
Positive
Positive
0051-04
Negative
Positive
0051-05
Negative
Positive
0051-07
Positive
0051-09
Evaluation of Rapid Test
• Specimen selection
o
Should represent the target population
For example:
For malaria Abs or Ags test, prefer specimens from population at
epidemic area
o
A appropriate number of positive and negative samples required
Recommend:
For sensitivity, to select at least 20 positive and 2 negative specimens
For specificity, to select at least 20 negative and 2 positive specimens
o
Should be collected, extracted and stored appropriately
Correct specimens handling and storage is critical for reliable evaluation
Evaluation of Rapid Test
• Proper Specimen dilution
Specimen
Diluent
Don’t (to avoid matrix effect)
Serum, plasma, whole blood
Diluted with negative specimen
Don’t dilute with assay diluent provided
in the kit
Fecal extraction specimen
Diluted with the extraction buffer
Don’t dilute with water or other buffer
Nasal extraction specimens
Diluted with the extraction buffer
Don’t dilute with water or other buffer
Genital extraction specimen
Diluted with mixed extraction
buffer at different ratio defined in
assay procedure
Don’t dilute with water or individual
extraction buffer
Urine specimen
Diluted with negative urine
Don’t dilute with water or other buffer
Use of proper specimen diluents are critical for reliable evaluation.
Evaluation of Rapid Test
• About discrepancy results
o
A 3rd party test with more sensitive detection technique should be
used to verify the discrepancy specimens
ELISA, chemiluminescence, or western blot test may be used to verify the
discrepancy by two rapid tests
o
Check if the specimen contains common interference factors:
RF, HAMA, ANA or others may interfere the results
Evaluation of Rapid Test
• Reference test
o Compare “Apple to Apple” - Use the same technology
RDTs for evaluation of RDTs; ELISA for evaluation of ELISA
o
Compare with the market leader


Errors of evaluation will arise if the reference test itself does not have
good sensitivity and specificity.
Reference kit has high false positive, the test kit becomes “false” negative
Reference kit has high false negative, the test kit becomes “false” positive
• Use of gold standard


Gold standard was usually developed many years ago
It may be lack of good sensitivity or specificity
• Reference standard
Commercial standards (such as NIBSC, BBI) are designed for the specific
technology (e.g. RDT or ELISA). A standard intended to be used with ELISA
may not be suitable with Rapid Test.
Standardization
• Concentration of analyte, such as PSA and Troponin I in specimens is
determined with different kits or machines
• The values from different kits or machines may be different
e.g.
Bio-Rad Liquicheck Cardiac Markers control level 1) contains 0.25ng/ml Troponin I.
The concentration of this control determined by different assays varies between
0.2-2.1 ng/ml
• When evaluating rapid tests with these specimens, one should take this
into consideration regarding the method to be used to determine the
value of the analyte.
Standardization against an international standard (WHO , NIST) is preferred
Common Questions in Product Evaluation
Questions
Potential Issues
Suggestion
T line is faint in Each company uses their own • As long as the line is present, one
comparison with the colloid gold formulation , so the should be confident in the test
competitor’s T line
gold color varies
result.
• Can ask 2nd person to verify the
weak color line
The test result of Rapid Specimen size is too small to Test with at least 20 specimens,
Test is different from that represent the true product ideally a panel of minimal 30
of competitor rapid test
performance.
positives and 70 negatives
Use competitor’s kit component in Only use the component provided
the comparison study
by the test kit, not to switch or
substitute
The LOD of the Rapid Test is • Compare the product with the
different from that of competitor’s
same LOD
• Send feedback to CTK for the test
with the same LOD
The result of Rapid Test is Bio-Rad control or other control is Test the control with the Rapid Test
negative on Bio-Rad or designed for quantitative test, such as well as with the market leader
other control
as ELISA, CLIA, might not suit for rapid test. “Compare Apple to
rapid test.
Apple”
Analysis of Sample Evaluation
Sample
ID
Company A
1
2
3
4
6
11
Negative
Negative
Negative
Negative
Negative
Negative
Negative
Negative
Negative
Negative
Negative
Negative
Negative
Negative
Strong positive
Strong positive
Weak positive
Strong positive
Strong positive
Strong positive
Strong positive
Strong positive
12
16
17
18
19
24
30
32
5
7
15
23
27
20
21
31
Company B
Company C
ELISA
Negative
Negative
Negative
Negative
Negative
Negative
Weak positive
Weak positive
Negative
Weak positive
Weak positive
Weak positive
Negative
Negative
Negative
Negative
Weak positive
Negative
Strong positive
Negative
Negative
Negative
N/A
Negative
Positive
N/A
N/A
Negative
N/A
Negative
N/A
Negative
N/A
N/A
Negative
Negative
N/A
N/A
positive
N/A
N/A
N/A
N/A
Positive
Leading brand
Negative
Negative
Negative
Negative
Negative
Negative
Negative
Negative
Negative
Negative
Negative
Negative
Negative
Negative
Strong positive
Strong positive
Weak positive
Strong positive
Strong positive
Strong positive
Strong positive
Negative
If we compare “Apple to tomato”, then
Company A kit has false negative .
Company C kit has inferior quality. If using
company C as reference, company A kit has
false negative or false positive. .
Tested against 22 specimens, company A kit
is similar as the leading kit
If only #31 specimen is tested, company A kit
has false positive. However the positive result
is verified by ELISA kit .
• Additional Information
6
Storage Temperature & Shelf Life
Reference:
Clinical and Laboratory Standard Institute. EP25-A. Evaluation of Stability of In
Vitro Diagnostic Reagents; Approved Guideline
Stability of an IVD Product
• Stability describes the ability of an IVD reagent to retain its
properties and performance over a specified time interval when
stored under specific conditions.
• The product stability is defined on the basis of ensuring that key
performance metrics are met within predefined acceptance criteria
throughout the claimed duration.
• IVD product stability is affected by external and internal variables,
such as storage conditions, handling, final product container system,
formulation.
Reference CLSI. EP25-A
Shelf Life of an IVD Product
• The Shelf life is the period of time until the expiration (expiry) date,
during which an IVD reagent in the packaging configuration provided
to the user maintains its stability under the storage conditions
specified by the manufacturer.
Reference CLSI. EP25-A
Temperature Outside the Specified Range Negatively
Affects the Shelf Life of an IVD Product
• Temperatures outside the range specified by the manufacturer
damage antibodies and antigens used in the IVD product, and
therefore reduce product performance.
• Based on the Arrhenius equation, the chemical reaction rate (e.g.
antibody degradation) may double for every 10˚C increase in
temperature.
Stability
Therefore, stability of an IVD product may decrease 2 times faster if
temperature is elevated 10˚C above recommended storage
temperature
Temperature
Reference CLSI. EP25-A
Temperature Outside the Specified Range Negatively
Affects the Shelf Life of an IVD Product
Rapid Test Shelf Life in Different Storage Condition
Rapid Test stable for
2-30⁰C
37⁰C
45⁰C
18 months ≈
9 months ≈
3 months
WHO qualifies a good rapid test that shows stability for 2 months at 45⁰C
storage (quote: WHO malaria rapid test evaluation program)
To Ensure IVD Product Quality and Reliability
• Store all components of an IVD product under conditions specified by
the manufacturer.
• Monitor and report temperature of storage areas daily.
• If product was exposed to temperatures outside the specified range:
o
Contact CTK technical service
o
Verify performance of the product
Temperature monitor report
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