1. OnSite Rapid Test Procedure Training
Part 4
CTK-MK-PPT-R0001 (4)

2. 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.

3. Training Contents Introduction Perform Assay Before, During, After 12
Perform Assay Before, During, After 3
Assaying with Accuracy 4
Troubleshooting 5
Product Evaluation
Additional Information
Storage Temperature & Shelf Life 6

4. 4
Tro uble sho otin g

5. 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:
Clinical findings
Confirmatory test

6. Limitation of Rapid Test
Is a qualitative or semi-quantitative device
Has detection limit
Analyte has genetic variation or strain variation
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
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 false-negative results on the P.f HRP-II antigen test

7. 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 anti-Hb Ab, leads to false negative result
Interference by other substances
Anticoagulants, or medicines present in the sample can inhibit the reaction, leads to false negative

8. 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 by other substances
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 defined time window
Increase of reaction time leads to false positive

9. 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
Use different C line system from the T line, such as rabbit antibody control line system, while the T line is mouse antibody system
Repeatedly invalid results
Defect devices
Check device for defective packaging
Verify device quality by external control
Inform supervisor and CTK

10. Troubleshooting Interpretation of assay result Problem
Potential Causes
Action
False positive
Wrong specimen type
Use correct specimen type
Specimen contains unusually high titers of lipids, RF, human anti-mouse Ab, anti-nuclear Ab or other inference
Spin specimen and get rid of lipid
Check for the presence of interference
Incorrect volume of specimens or buffer
Strictly follow IFU procedure
Using components from different kit
Don’t use component from different kit
Read results exceeded defined in IFU
Using distilled water or other inappropriate solution as external control specimen
Use clinical specimens or provided control as external control
False negative
Read result less or exceed defined in IFU
Specimen is collected too early
Collect specimen few hours, days or weeks late
Analyte present below the LOD
Use more sensitive detection methods
IFU: Instruction for Use

11. Troubleshooting External Control Storage condition Problem
Potential Causes
Action
External control fails to show accurate results
Wrong procedure to reconstitute control
Strictly follow control IFU
Control is not stored as required
Store control properly. Return control to its storage condition immediate after use, if the control is for multiple use
Control is expired
Use only valid control
Storage condition
Problem
Potential Causes
Action
Storage temperature exceeds recommended range
Storage does not have A/C system to control temperature
Run external control to verify kit quality
Kit is frozen
External temperature is too cold during transportation or accidently keep in freezer
Bring up the kit into room
temperature before testing,
Verify the kit qualify with external
control

12. 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
Limitation
of Test
Review Manufacture Instruction
Test with
Alternative Methods
External Control
Retest with
Different Lot/Device
Report to CTK

13. Trouble shooting – Follow the Correct Procedure!
More than 50% of complaints received are caused by incorrect procedure
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
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!

14. Complaint Handling Procedure
Customer Complaints
Recorded and Classified the Complaints
by Customer Service & QA
General Complaints
(Not Products Related)
Product Complaints
Check Procedure
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

15. 5
Pro duc t Eval uati on

16. 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 Sensitivity
High Specificity
Increased desire to avoid false positives if the illness is rare
or treatment is burdensome
to the patient

17. Evaluation of Rapid Test Collection, Extraction, Storage, Dilution
Evaluation protocol
Specimen
Collection, Extraction, Storage, Dilution
Compare
“Apple to Apple”
OnSite Rapid Test
Competitor Rapid Test
Results
Results
Evaluation
Agree
Disagree: resolve discrepant results
Compare
to 3rd party test
Conclusion

18. Evaluation of Rapid Test
Performance on BBI Mixed Titer Performance Panel 08454
RDT
OnSite
competitor
ELISA
IgM
IgG
Positive
Negative
Step1
Comparison with
competitor RDT
Step2
Resolve difference
By ELISA
Conclusion:
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

19. Evaluation of Rapid Test
Specimen selection
Should represent the target population
A appropriate number of positive and negative samples required
Should be collected, extracted and stored appropriately
For example:
For malaria Abs or Ags test, prefer specimens from population at epidemic area
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
Correct specimens handling and storage is critical for reliable evaluation

20. 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
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
Use of proper specimen diluents are critical for reliable evaluation.

21. Evaluation of Rapid Test
About discrepancy results
A 3rd party test with more sensitive detection technique should be used to verify the discrepancy specimens
Check if the specimen contains common interference factors:
ELISA, chemiluminescence, or western blot test may be used to verify the discrepancy by two rapid tests
RF, HAMA, ANA or others may interfere the results

22. Evaluation of Rapid Test
Reference test
Compare “Apple to Apple” - Use the same technology
Compare with the market leader
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
RDTs for evaluation of RDTs; ELISA for evaluation of ELISA
Errors of evaluation will arise if the reference test itself does not have good sensitivity and specificity.
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.

23. 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 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

24. Common Questions in Product Evaluation
Potential Issues
Suggestion
T line is faint in comparison with the competitor’s T line
Each company uses their own colloid gold formulation , so the gold color varies
As long as the line is present, one should be confident in the test result.
Can ask 2nd person to verify the weak color line
The test result of Rapid Test is different from that of competitor rapid test
Specimen size is too small to represent the true product performance.
Test with at least 20 specimens, ideally a panel of minimal 30 positives and 70 negatives
Use competitor’s kit component in the comparison study
Only use the component provided by the test kit, not to switch or substitute
The LOD of the Rapid Test is different from that of competitor’s
Compare the product with the same LOD
Send feedback to CTK for the test with the same LOD
The result of Rapid Test is negative on Bio-Rad or other control
Bio-Rad control or other control is designed for quantitative test, such as ELISA, CLIA, might not suit for rapid test.
Test the control with the Rapid Test as well as with the market leader rapid test. “Compare Apple to Apple”

25. Analysis of Sample EvaluationID
Company A
Company B
Leading brand
Company C
ELISA 1
Negative
N/A 2 3
Positive 4 6 11 12
Weak positive 16 17 18 19 24 30 32 5
Strong positive 7 15
positive 23 27 20 21 31
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 .

26. Add itio nal Info rma tion Storag e Temp eratur e & Shelf Life6
Add itio nal Info rma tion Storag e Temp eratur e & Shelf Life
Reference:
Clinical and Laboratory Standard Institute. EP25-A. Evaluation of Stability of In Vitro Diagnostic Reagents; Approved Guideline

27. 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

28. 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

29. 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.
Therefore, stability of an IVD product may decrease 2 times faster if temperature is elevated 10˚C above recommended storage temperature
Temperature
Stability
Reference CLSI. EP25-A

30. Temperature Outside the Specified Range Negatively Affects the Shelf Life of an IVD Product

31. Rapid Test Shelf Life in Different Storage Condition
Rapid Test stable for
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)

32. 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:
Contact CTK technical service
Verify performance of the product
Temperature monitor report

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