TestLine RFM096 Is Am Auto Immune Disease Instruction Manual
- June 13, 2024
- TestLine
Table of Contents
- Document Record
- Intended Purpose
- Introduction
- Test Principle
- Materials Provided
- Other Material Required for Test Performance
- Storage and Stability
- Preparation of Reagents
- Preparation of Samples
- Working Schedule
- Quality Control
- Results Interpretation
- Analytical Performance
- Specificity and Sensitivity
- Trueness (bias)
- Precision: Repeatability – Intra-assay (within run
- Precision: Reproducibility – Inter-assay (between-run)
- Accuracy
- Analytical sensitivity – limits of detection and quantitation
- Measuring Range
- Linearity
- Hook effect
- Comparison with the reference method
- Table 3 Analytical Performance
- Interference
- Cross-reactivity
- Clinical Performance
- Safety Precautions
- Procedural Notes
- References
- IFU Symbols
- Summary of EIA RF IgM Protocol
- CUSTOMER SERVICE
- References
- Read User Manual Online (PDF format)
- Download This Manual (PDF format)
TestLine RFM096 Is Am Auto Immune Disease
Document Record
Revision No. | Version No. | Revision Description |
---|---|---|
ZM01566 ZM01546 | 25 | Revision according to IVDR requirements Correction of |
sensitivity, specificity, precision values
ZM01876| 26| Correction of the chapter Antigen Used
Intended Purpose
The immunoenzymatic assay is intended for the diagnosis of rheumatoid arthritis using IgM antibodies to rheumatoid factor in human serum or plasma in the general population. The semi-quantitative and quantitative manual assay is designed for professional use in a laboratory.
Introduction
Rheumatoid arthritis (RA) is an autoimmune disease which is characterised by
chronic inflammation of joint synovium of eroding cartilage and bones. The
pathology often leads to the destruction of the joints. RA affects more than 5
million people. The disease most commonly starts between 30 and 50 years of
age. Women are affected three times as often as men. The cause of RA is still
not completely known.
Determination of rheumatoid factors (RF) is the most commonly used laboratory
test in diagnostics of the disease. RF are immunoglobulins with an antibody
activity against the C-terminal part of the constant region of the human IgG
heavy chain, the IgG Fc fragment. They are found in the serum of RA patients
although they may be also found at lower frequency in several other diseases,
e.g. Sjögren´s syndrome, SLE or bacterial endocarditis. Assays based on
agglutination, turbidimetry, nephelometry or ELISA are used for RF screening.
ELISA method enables quantitative determination of IgA, IgG and IgM RF with
high sensitivity and specificity. IgM, IgG and IgA RF can be present in serum
individually as well as all together in dependence on clinical parameters and
disease activity. There is a proofed correlation between IgA and IgG RF levels
and disease prognosis.
Test Principle
The kit is intended for detection of specific IgM antibodies in a sample by
means of a sandwich type of the EIA method (i.e. a solid phase coated with
specific antigen – antibody from the analysed sample – labelled antibody). The
labelled antibody (conjugate) is an animal immunoglobulin fraction to human
IgM conjugated with horseradish peroxidase. Peroxidase activity is determined
in the test by a substrate containing TMB. Positivity is indicated when blue
colour appears; after stopping solution has been added, blue changes to
yellow. The yellow colour intensity is measured by a photometer at 450 nm, and
it is proportional to the concentration of specific IgM antibodies in the
sample.
Antigen Used
Purified immunoglobulin IgG
Materials Provided
| Microtitre Plate| 1 pc
---|---|---
| coated with antigen, 12 x 8 wells in bag with desiccant|
| **Negative Control (Calibrator
- 5 U/ml| 1 × 2 ml
| Solution containing no specific human antibodies, ready to use|
| CUT-OFF (Calibrator 2) 20 U/ml| 1 × 3 ml
| Solution containing specific human antibodies in cut- off concentration, ready to use
| Positive Control (Calibrator - 80 U/ml| 1 × 2 ml
| Solution containing specific human antibodies, ready to use
| Calibrator 4 (320 U/ml)| 1 × 2 ml
| Solution containing specific human antibodies, ready to use
| Conjugate| 1 × 15 ml
Solution containing peroxidase labelled animal immunoglobulin to human IgM, ready to use|
| Sample Diluent 2| 1 × 105 ml
Buffer with protein stabilisers, ready to use|
| TMB-Complete 2| 1 × 15 ml
| Chromogenic substrate solution containing TMB/H2O2, ready to use|
| Wash Solution| 1 × 75 ml
| 20× concentrated buffer|
| Stop Solution| 1 × 15 ml
| Acid solution, ready to use|
**| Instructions for use| 1 pc
Other Material Required for Test Performance
Single and multichannel pipettes Disposable tips Microplate washer Timer Incubator (37 °C) Microplate reader
Storage and Stability
Store the kit at +2 °C to +8 °C. Do not freeze. If the kit is stored as described, the labelled expiration date is valid. The expiration date is indicated on the package. The opened kit should be used within three months.
Sample Preparation and Storage
Samples listed in the intended use may be used for the examination. The
following human body liquids can be used for testing: serum and citrate
plasma. Anticoagulants in the plasma (except for citrate) as well as
bacterially contaminated, haemolytic or chylous samples can affect the test
results. A coagulating blood collection tube is recommended for serum
collection. A citrated plasma collection bag is recommended for plasma
collection. Other types of plasma (EDTA, heparin) may be used but they are not
recommended since anticoagulants may affect the test result.
Follow the manufacturer’s instructions when using commercial or other
specially modified samples. Clinical samples collected within standard medical
procedures into standardized tubes are ready for immediate use. Centrifugation
or other separations are not required. The examined samples can be stored at
+2 °C to +8 °C for a maximum of 1 week.
Preparation of Reagents
Dilute the Wash Solution 1:20 (1 part of solution and 19 parts of distilled
water); e.g. 75 ml of the concentrated Wash Solution + 1425 ml of distilled
water.
Salt crystals might develop in the bottle with the concentrated Wash Solution.
Prior to use, it is necessary to dissolve the crystals by warming the bottle
in a water bath. The diluted Wash Solution is stable at +2 °C to +8 °C for one
week. The Controls and the Calibrators are supplied ready to use, do not
dilute further! The Conjugate is supplied ready to use, do not dilute further!
TMB-Complete is a one-component chromogenic substrate solution ready to use,
do not dilute further!
Interchangeability of reagents
The Sample Diluent, TMB-Complete and the Avidity Solution are interchangeable in EIA kits of TestLine Clinical Diagnostics s.r.o., provided they have the identical numeric marking (e.g. Sample Diluent 2, Sample Diluent 3, etc.). The Stop Solution and the Wash Solution are universal in all kits.
Preparation of Samples
Mix gently the Sample Diluent prior to use.
Dilution of sera and plasma samples Dilute well mixed samples 1:101 with the
Sample Diluent: E.g.: 10 µl of sample + 1 ml of the Sample Diluent Mix well.
Assay Procedure
Allow all reagents to come to room temperature and mix well. If you do not use
a whole microplate, return unnecessary strips into the bag with desiccant.
Seal the bag tightly and store at +2 °C to +8 °C. Keep dry!
-
Dispense the controls (calibrators) and the diluted samples according to the working schedule.
Semiquantitative evaluation in Index of Positivity (IP)
− Leave A1 well empty (blank).
− Pipette 100 µl of the Negative Control (Calibrator 1) into 1 well.
− Pipette 100 µl of CUT-OFF (Calibrator 2) into 2 wells.
− Pipette 100 µl of the Positive Control (Calibrator 3) into 1 well.
− Pipette 100 µl of the diluted samples (see Chapter Preparation of Samples) into the other wells.
Quantitative evaluation in Units U/ml
− Leave A1 well empty (blank).
− Pipette 100 µl of the Negative Control (Calibrator 1) into 1 well.
− Pipette 100 µl of CUT-OFF (Calibrator 2) into 2 wells.
− Pipette 100 µl of the Positive Control (Calibrator 3) into 2 wells.
− Pipette 100 μl of the Calibrator 4 into 2 wells.
− Pipette 100 µl of the diluted samples (see Chapter Preparation of Samples) into the other wells. -
Cover the microplate with the lid and incubate at 37 °C for 30 minutes.
-
Aspirate the content of the wells and wash 5× with the working strength Wash Solution. Fill the wells up to the edge. Finally, tap the inverted microplate thoroughly on an absorbent paper to remove solution remnants.
-
Pipette 100 µl of the Conjugate into all wells except A1 well.
-
Cover the microplate with the lid and incubate it at 37 °C for 30 minutes.
-
Aspirate the content of the wells and wash 5× with the working strength Wash Solution. Fill the wells up to the edge. Finally, tap the inverted microplate thoroughly on an absorbent paper to remove solution remnants.
-
Pipette 100 µl of TMB-Complete into all wells. Avoid contamination – see Chapter Procedural
Notes. -
Cover the microplate with the lid and incubate at 37 °C for 30 minutes. Keep out of light.
-
Stop the reaction by adding 100 µl of the Stop Solution in the same order and intervals as the substrate was added.
-
Read the colour intensity in wells against blank (A1 well) using photometer set to 450 nm. The absorbance should be read within 30 minutes after stopping the reaction.
Working Schedule
Semiquantitative evaluation
Index of Positivity (IP)
BL| Blank (empty well)|
---|---|---
NC| 100 µl|
CO| 100 µl
PC| 100 µl
TS 1-x| 100 µl
Quantitative evaluation
Units U/ml
BL| Blank (empty well)|
---|---|---
Cal1| 100 µl|
Cal2| 100 µl
Cal3| 100 µl
Cal4| 100 µl
TS 1-x| 100 µl
Quality Control
Test is valid if:
The absorbance of blank is lower than 0.150.
BLANK < 0.150
The absorbance of the Negative Control (Calibrator 1) is lower than half of
the mean absorbance of CUT‒OFF (Calibrator 2).
The mean absorbance of CUT-OFF (Calibrator 2) is within a range of 0.150 –
0.900.
The absorbance of the Positive Control (Calibrator 3) is 1.5-fold higher than
the mean absorbance of CUT‒OFF (Calibrator 2).
The absorbance of the Calibrator 4 is higher than the absorbance of the
Positive Control (Calibrator 3).
Results Interpretation
Calculation of Index of Positivity (IP)
Divide the absorbance of a tested sample by the mean absorbance of CUT-OFF
measured in the same test run
Interpretation of the test results is described in the table (Table 1).
Table 1 Interpretation of test results
Index of Positivity (IP) | Evaluation |
---|---|
lower than 0.9 | negative |
0.9 to 1.1 | borderline |
higher than 1.1 | positive |
Examination of borderline samples, i.e. samples with Index of Positivity from 0.9 to 1.1, should be repeated from a new sample collected after 2 to 6 weeks regarding to the disease specifics.
Quantitative evaluation in Units (U/ml)
Construct a calibration curve by plotting the concentration (X) of the
calibrators in U/ml against the corresponding absorbance (Y). Construct the
calibration curve by single point cross connection. Read the values of
antibody level (U/ml) in samples from the calibration curve. Interpretation of
the quantitative test results is described in the table (Table 2).
Table 2 Quantitative interpretation in Units (U/ml)
Antibody level (U/ml) | Evaluation |
---|---|
lower than 18 | negative |
18 to 22 | borderline |
higher than 22 | positive |
Examination of borderline samples should be repeated from a new sample
collected after 2 to 6 weeks regarding to the disease specifics.
Serological finding can be interpreted only in the context of results of other
laboratory tests and patient clinical picture.
Analytical Performance
Specificity and Sensitivity
Specificity was determined in the panel of negative samples. Sensitivity was determined in the panel of positive samples. The number of samples tested and the results obtained are described in the table (Table 3).
Trueness (bias)
Trueness is the closeness of agreement between the average value obtained from a large number of measurement results and the reference value. Its measure is bias. The nature of the method does not allow quantitative determination of bias (and thus the trueness). The trueness of the method is ensured by clinical parameters such as sensitivity and specificity, comparison with the reference method and batch continuity. The obtained results are described in the table (Table 3).
Precision: Repeatability – Intra-assay (within run
The precision is defined as the closeness of agreement between measured values obtained by replicate measurements on the same object under specified conditions. The Intra-assay repeatability is expressed as agreement level among sample replicates within a run of the assay (in one batch). The obtained results are described in the table (Table 3).
Precision: Reproducibility – Inter-assay (between-run)
Reproducibility is a measure of precision under a defined set of conditions which include the interassay, expressed as agreement level among sample replicates within runs of the assay in one batch. The obtained results are described in the table (Table 3).
Accuracy
Accuracy is defined as the closeness of agreement between the measured value and the reference value. It is expressed as an achievable measure of the combined uncertainty. The obtained results are described in the table (Table 3).
Analytical sensitivity – limits of detection and quantitation
The analytical sensitivity is the maximum binary dilution of CUT-OFF or international standard samples, respectively, giving absorbance significantly different from the background. The value is expressed as an index of positivity and/or a concentration in units. This value is a minimum limit of detection and quantification. The obtained results are described in the table (Table 3).
Measuring Range
The measuring range of the kit lies between values where the lower limit is determined by the analytical sensitivity value and the upper limit depends on the measuring capability of the equipment used.
Linearity
The linearity is the ability of the method to obtain final values proportional (directly or after mathematical transformation) to the concentration of analyte in the sample; it is expressed as the range in which the method provides linear results. The obtained results are described in the table (Table 3).
Hook effect
Hook effect is an immunological phenomenon that causes falsely low results in the presence of an excess amount of analyte. Its presence is detected by serial dilution of a highly positive sample (Table 3).
Comparison with the reference method
Comparison with the reference method was performed. The results of both methods are comparable, considering the differences of both methods and completely meeting the requirements if the agreement in the classification of the samples is at least 90% (Table 3).
Table 3 Analytical Performance
Parameter | Value |
---|---|
Sensitivity (n 86) | 95.12% |
Specificity (n 88) | 97.73% |
Trueness (bias) | N/A |
Precision: Repeatability | 3.79% |
Precision: Reproducibility | 3.82% |
Accuracy | 6.31% |
Analytical sensitivity | IP 0.03 |
0.63 U/ml
Linearity interval| IP 0.51–4.37
12.13–138.99 U/ml
Hook effect| Not observed
Comparison with the reference method| At least 90%
N/A – not applicable
Interference
Two samples (one negative plasma pool and one positive plasma pool) were spiked with potentially interfering substances. Results of interference testing are shown in the table (Table 4).
Table 4 Interference Results
Interfering substance | The result was not affected up to concentration: |
---|---|
Bilirubin | 0.4 mg/ml |
Triacylglycerols | 20 mg/ml |
Haemoglobin | 5 mg/ml |
Cross-reactivity
The assay was evaluated for potential cross-reactivity using samples positive for selected pathogens and factors. Results of testing are shown in the table (Table 5).
Table 5 Results of Cross-Reacting Pathogens or Factors
Category | (n) | Positive result |
---|---|---|
EBV VCA | 6 | 0 |
CMV | 4 | 0 |
Toxoplasma gondii | 10 | 0 |
HSV 1+2 | 6 | 0 |
Rubella virus | 5 | 0 |
Borrelia spp. | 10 | 1 |
Total | 41 | 1 |
Clinical Performance
Diagnostic specificity and diagnostic sensitivity
Diagnostic specificity was determined in the panel of negative samples. Diagnostic sensitivity was determined in the panel of positive samples. The number of samples tested and the results obtained are shown in the table (Table 6).
Positive and negative predictive value
A positive predictive value is probability that a person is actually affected by infection if the result was positive. A negative predictive value is probability that a person is actually healthy if the result was negative. The results obtained are shown in the table (Table 6).
Likelihood ratio of the kit
The likelihood ratio of the kit for a positive test is the ratio of probability that an individual from affected population is diagnosed as positive by the test and probability that a healthy individual is misdiagnosed as positive.
The likelihood ratio of the kit for a negative test is the ratio of probability that an individual from affected population is misdiagnosed as negative by the test and probability that a healthy individual is diagnosed as negative. The results obtained are shown in the table (Table 6).
Expected values in population
Expected values in population are established based on the value results in a file of samples declared as negative and a file of samples declared as positive for the presence of specific antibodies. The results obtained are shown in the table (Ta
Table 6 Clinical performance
Parameter | Value | 95% Confidence Interval (CI) |
---|---|---|
Diagnostic sensitivity (n 86) | 95.12% | 87.98–98.66% |
Diagnostic specificity (n 88) | 97.73% | 92.03–99.72% |
Positive predictive value (n 86) | 97.50% | 91.26–99.70% |
Negative predictive value (n 88) | 95.56% | 89.01–98.78% |
Likelihood ratio of the kit for a positive test | 41.854 | – |
Likelihood ratio of the kit for a negative test | 0.050 | – |
Expected values in healthy population | IP 0.26 | IP 0.23–0.29 |
Expected values in affected population | IP 2.29 | IP 2.12–2.46 |
Safety Precautions
The kit is intended for in vitro diagnostic use only.
The sera used for controls were tested and found to be negative for HIV 1 and
HIV 2, HBsAg, HCV, TPHA. In spite of this fact, they still need to be handled
as potentially infectious materials.
Some reagents contain the toxic component sodium azide or gentamicin, but in
very low concentrations. Avoid contact with skin.
The Stop Solution contains diluted acid solution. Avoid contact with eyes and
skin.
It is necessary to observe the local safety rules and regulations.
First aid
In case of contact with eyes, flush with copious amount of water and seek
medical assistance. In case of contact with skin and clothing, remove all the
contaminated clothes. Wash the skin with soap and plenty of running water. In
case of contact with solutions containing plasma or clinical samples,
disinfect the skin. In case of accidental ingestion, flush the mouth with
drinking water and seek medical assistance.
Remnants disposal
All the materials used for performing the test must be treated as potentially
infectious due to the contact with biological materials. Therefore they need
to be disposed together with biological waste.
Expired kit disposal
Disassemble the kit and dispose the components as biological material. Discard
the packaging material as required by local regulations.
Procedural Notes
In order to obtain reliable results, it is necessary to strictly follow the
Instructions for Use. Always use clean preferably disposable tips and
glassware.
Microtitre Plate – in order to prevent water condensation on the surface of
the microplate, always allow the bag with the microplate to warm up to room
temperature before opening.
Wash Solution – use high quality distilled water for preparing the working
strength Wash Solution. Washing procedure – keep to the prescribed number of
wash cycles and fill the wells to the upper edge. The soak time (i.e. interval
between two different wash cycles during which the wells stay filled up with
the Wash Solution) should be approx. 30–60 sconds.
TMB-Complete – the vessel used for multichannel pipetting should not be used
for other reagents.
Do not return the surplus TMB-Complete from the pipetting vessel into the
vial.
Non-reproducible results might be caused by improper methodology as following:
− insufficient mixing of reagents and samples before use
− improper replacement of vial caps
− using the same tip for pipetting different reagents
− reagent exposure to excessive temperature; bacterial or chemical
contamination
− insufficient washing or filling of the wells (the wells should be filled to
the upper edge), improper aspiration of Wash Solution remnants
− contamination of the well edges with Conjugate or samples
− using reagents from different kit lots
− contact of reagents with oxidants, heavy metals and their salts
Technical limitation of samples
Materials of a human origin from donor population listed in the intended use
were used for manufacture and development of the kit. Kits are intended for
use in general population, unless otherwise stated.
When using samples from other specific populations (comorbid,
immunocompromised, pregnant, paediatric population), the risk of a specific
effect on the result of the applied test due to e.g.interference or cross-
reactivity should be considered in the context of expert knowledge and current
scientific knowledge.
Other notes
The kit might be used for sequential examinations. When preparing working
strength solutions, use only the amount of reagents needed for the analysis.
The kit might be used in all types of automatic EIA analysers. If necessary,
TestLine Clinical Diagnostics s.r.o. can offer a certified modification of the
Instructions for Use for the specific type of analyser.
The producer cannot guarantee that the kit will function properly if the assay
procedure instructions are not strictly adhered to.
References
- Allen C, Elson CJ, Scott DGI, Bacon PA, Bucknall RC. IgG anti-globulins in rheumatoid arthritis and other arthritis’s: relationship with clinical features and other parameters. Ann Rheum Dis. 1981. 40: 127–131.
- Amason JA, Johnsson TH, Brekkan A, Sigurjohnsson K, Valdimarsson H. Relation between bone erosions and rheumatoid factor isotypes. Ann Rheum Dis. 1987. 46: 380–384.
- Bampton JLM, Cawston TE, Kyle MV, Hazleman BL. Measurement of rheumatoid factors by an enzyme linked immunosorbent assay (ELISA) and comparison with other methods. Ann Rheum Dis. 1985. 44: 14–19.
- Dunne JV, Carson DA, Spiegelberg HL, Alspaugh MA, Vaughan JH. IgA rheumatoid factor in the serum and saliva of patients with rheumatoid arthritis and Sjörgen’s syndrome. Ann Rheum Dis. 1979. 38: 161–166.
- Geirson AJ, Sturfelt G, Truedsson L. Clinical and serological features of severe vasculitis in rheumatoid arthritis: prognostic implications. Ann Rheum Dis. 1987. 46: 727–733.
- Gioud-Paquet M, Auvinet M, Raffin T et al. IgM rheumatoid factor (RF), IgA RF, IgE RF, and IgG RF detected by ELISA in rheumatoid arthritis. Ann Rheum Dis. 1987. 46: 65–71. 7. Kallerup HE, Egeskjold EM, Graudal H. IgG, IgM and IgA rheumatoid patients by an indirect immunofluorescence method. Scand J Rheumatol. 1979. 8: 1–9.
- Witte T, Hartung K, Sachse C, Matthias T, Fricke M, Kalden JR, Lakomek HJ, Peter HH, Schmidt RE. Rheumatoid factors in systemic lupus erythematosus: Association with clinical laboratory parameters. Rheumatol Int. 2000. 19: 107–111.
IFU Symbols
**** | Temperature limitation |
---|---|
**** | Keep dry |
**** | Expiry date |
**** | Lot number |
**** | Manufactured by |
**** | Consult instructions |
**** | Catalogue number |
**** | Number of tests |
**** | In vitro diagnostic medical device |
Summary of EIA RF IgM Protocol
Step No.| Symbol| Test steps|
---|---|---|---
1| | Dilute samples serum/plasma 1:101 (10 µl + 1 ml)|
2| | Pipette Controls (Calibrators) and diluted samples – 100 µl Blank = empty
well|
3| | Incubate at 37 °C for 30 min|
4| | Aspirate and wash the wells 5|
5| | Pipette Conjugate – 100 μl Blank = empty well|
6| | Incubate at 37 °C for 30 min|
7| | Aspirate and wash the wells 5×|
8| | Pipette Substrate (TMB-Complete) – 100 µl Including blank|
9| | Incubate at 37 °C for 30 min|
10| | Pipette Stop Solution – 100 µl Including blank|
11| | Read colour intensity at 450 nm|
CUSTOMER SERVICE
RFM096
96
Kit for professional use
Testline Clinical Diagnostics s.r.o.
– Krizikova 68, 612 00 Brno, Czech Republic
Tel.: +420 541 248 311
FAX: +420 541 243 390
E-mail: info@testlinecd.com
www.testlinecd.cz
www.testlinecd.co