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

1

•Introduction

2

•Perform Assay Before, During, After

3

•Assaying with Accuracy

4

•Troubleshooting

5• Product Evaluation

6• Additional Information Storage Temperature & Shelf Life

4

•Troubleshooting

• 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

• Analyte has genetic variation or strain variation

Variation ExampleIsotype 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

limitation ExampleTroponin 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


• False Negative

It means a test result indicates no presence of analyte (the result is negative), when compared to reference test.

Causes ExplanationLimit 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


• False Positive

It means a test result indicates presence of analyte (the result is positive), when compared to reference test.

Causes ExplanationCross 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

Problem Potential Causes ActionNo 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

Troubleshooting

• Internal control line (C line)

Problem Potential Causes ActionFalse 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 •Strictly follow IFU procedure Using distilled water or other inappropriate solution as external control specimen

•Use clinical specimens or provided control as external control

False negative Wrong specimen type • Use correct specimen type Incorrect volume of specimens or buffer • Strictly follow IFU procedureRead 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

Troubleshooting

IFU: Instruction for Use

• Interpretation of assay result

Problem Potential Causes ActionExternal 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

Troubleshooting

• External Control

Problem Potential Causes ActionStorage 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

• Storage condition

Troubleshooting Flow Chart

Unexpected Results

External Control

Retest with Different Lot/Device

Report to CTK

Procedure Limitation of Test

Test with Alternative Methods

Storage & Test Environment

Review Manufacture Instruction

Specimen Collection, Extraction & Storage

Review Procedure and Repeat Assay with a New Device

Repeated Unexpected Results

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!

QA & Product Manager Reply to Customer

Complaint Handling Procedure

Customer Complaints

General Complaints(Not Products Related)

Recorded and Classified the Complaints by Customer Service & QA

Investigated by QARecord, Review, Inspection, Reference

Product Complaints

Evaluated and Processed by Customer Service

Corrective and Preventive Action if Necessary

Customer Service Reply to Customer

Reviewed and Closed by QA

Corrective and Preventive Action if Necessary

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

Increased desire to avoid false positives if the illness is rare or treatment is burdensome

to the patient

High SensitivityHigh Specificity


• Evaluation protocol

SpecimenCollection, Extraction, Storage, Dilution

OnSite Rapid Test Competitor Rapid Test

Results Results

Evaluation• Agree• Disagree: resolve discrepant results

Conclusion

Compare “Apple to Apple”

Compare to 3rd party test

Performance on BBI Mixed Titer Performance Panel 08454

RDTOnSite

RDT competitor

ELISA

IgM IgG IgM IgG IgM IgG

0051-01 Positive Positive Positive Positive Positive Positive

0051-04 Negative Positive Negative Negative Negative Positive

0051-05 Negative Positive Negative Positive Negative Positive


0051-09 Positive Positive Positive Positive Negative Positive




0051-14 Negative Negative Negative Negative Negative Negative


0051-16 Positive Positive Positive Positive Negative Positive







Step1Comparison with competitor RDT

Step2Resolve differenceBy 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


• Specimen selectiono Should represent the target population

o A appropriate number of positive and negative samples required

o Should be collected, extracted and stored appropriately

Recommend:For sensitivity, to select at least 20 positive and 2 negative specimensFor specificity, to select at least 20 negative and 2 positive specimens

Correct specimens handling and storage is critical for reliable evaluation

For example:For malaria Abs or Ags test, prefer specimens from population at epidemic area


• Proper Specimen dilution

Use of proper specimen diluents are critical for reliable evaluation.

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


• About discrepancy resultso A 3rd party test with more sensitive detection technique should be

used to verify the discrepancy specimens

o 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


• Reference testo Compare “Apple to Apple” - Use the same technology

o 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.

• 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

Standardization

Common Questions in Product Evaluation

Questions Potential Issues SuggestionT 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”

SampleID

Company A Company B Leading brand

Company C ELISA

1 Negative Negative Negative N/A2 Negative Negative Negative Negative3 Negative Negative Negative Positive4 Negative Negative Negative N/A

6 Negative Negative Negative N/A

11 Negative Negative Negative Negative

12 Negative Negative Weak positive N/A

16 Negative Negative Weak positive Negative17 Negative Negative Negative N/A18 Negative Negative Weak positive Negative19 Negative Negative Weak positive N/A24 Negative Negative Weak positive N/A

30 Negative Negative Negative Negative32 Negative Negative Negative Negative5 Strong positive Strong positive Negative N/A7 Strong positive Strong positive Negative N/A15 Weak positive Weak positive Weak positive positive23 Strong positive Strong positive Negative N/A27 Strong positive Strong positive Strong positive N/A20 Strong positive Strong positive Negative N/A21 Strong positive Strong positive Negative N/A31 Strong positive Negative Negative Positive

If only #31 specimen is tested, company A kit has false positive. However the positive result is verified by ELISA kit .

Company C kit has inferior quality. If using company C as reference, company A kit has false negative or false positive. .

If we compare “Apple to tomato”, then Company A kit has false negative .

Tested against 22 specimens, company A kit is similar as the leading kit

Analysis of Sample Evaluation

6

• Additional Information 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.


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

Stab

ility


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

Rapid Test Shelf Life in Different Storage Condition

2-30⁰C 37⁰C 45⁰C

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)

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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Contact Info.:10110 Mesa Rim Rd. San Diego, CA 92121 USAEmail Address: [email protected] Telephone: 1 (858) 457-8698Fax: 1 (858) 535-1739

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