for the standard of highest concentration (D1) then the concentration of IgA in the test specimen can be determined directly by interpolating ∆A of the test

 

Initial RF positivity has been a sensitive predictor for later

joint destruction. Quantified measurement of initial RF

level and especially repeated measurements of RF at regular

intervals seems to add significantly to the prognostic value

of RF in distinguishing between progressive and nonprogressive disease in early RA.

QUANTIA-RF is a turbidimetric immunoassay for

quantitative detection of rheumatoid factors of the IgM

class.

Reagent

1. QUANTIA-RF activation buffer (R1): Ready-to-use

buffer.

2. QUANTIA-RF latex reagent (R2): Ready-to-use uniform

suspension of polystyrene latex particles coated with

suitably modified Fc fraction of human IgG.

3. QUANTIA-RF calibrator: Lyophilized preparation of RF

positive serum, which is equivalent to stated amount

of RF on IU/mL basis, when hydrated appropriately.

The QUANTIA-RF calibrator is traceable to the WHO,

International Reference Preparation of Rheumatoid

Arthritis Serum.

Each batch of reagents undergoes rigorous quality

control at various stages of manufacture for its specificity,

sensitivity and performance.

Reagent Storage and Stability

1. Store the reagents at 2 to 8°C. Do not freeze.

2. The shelf-life of the reagent, activation buffer and

the calibrator is as per the expiry date mentioned

on the respective vial labels.

3. The reconstituted QUANTIA-RF calibrator is stable

for 7 days at 2 to 8°C and 48 hours at 25 to 30°C (RT).

4. The working reagent for QUANTIA-RF can be prepared

by mixing R2 and R1 in the ratio 1:5.

5. The mixed stability of the working reagent (R1+ R2) is

7 days when stored at 2 to 8°C.

Principle

QUANTIA-RF is a turbidimetric immunoassay for the

determination of rheumatoid factors and is based on the

principle of agglutination reaction. The test specimen

is mixed with QUANTIA RF latex reagent (R2) and

activation buffer (R1) and allowed to react. Presence of RF

in the test specimen results in formation of an insoluble

complex resulting in an increase in turbidity, which is

measured at wavelength 505 to 578 nm. The increase in

turbidity corresponds to the concentration of RF in the test

specimen.

Note

1. In vitro diagnostic reagent for laboratory and

professional use only. Not for medicinal use.

2. All the reagents derived from human source have

been tested for HBsAg and HIV antibodies and are

found to be non-reactive. However, handle the

material as if infectious.

3. Reagents contain 0.1% sodium azide as preservative.

Avoid contact with skin and mucosa. On disposal,

flush with large quantities of water.

4. The reagents can be damaged due to microbial

contamination or on exposure to extreme temperatures. It is recommended that the performance

of the reagents be verified using known controls

periodically.

5. Gently mix the QUANTIA-RF latex reagent well

before use to disperse the latex particles uniformly to

improve test performance.

6. The working reagent should be mixed gently.

7. Do not use vortex mixers for mixing. Gently mix the

reagents and samples during test procedures.

8. As the reagents within lots have been matched,

reagents from different lots must not be interchanged.

9. Calibrators of different manufacturers must not be

used with QUANTIA-RF reagents.

10. The calibration curve must be validated periodically

with known controls.

11. The QUANTIA-RF assay is recommended only

for analyzers with cuvette mode. Though any

semiautomated analyzer with appropriate programing

facility can be used, for best results it is recommended

to use; Quantiamate analyzer. Fully automated

Diagnostic Immunology 719

analyzers may be used, provided the reagent has been

standardized on the system.

12. The procedure mentioned here is based on a

minimum reading volume of 500 µL (0.5 mL). In case

of instruments where minimum volume required for

reading absorbance is 1.0 mL, use double the quantity of reagents and samples mentioned in the test

procedure.

Specimen Collection and Preparation

No special preparation of the patient is required prior to

specimen collection by approved techniques.

Only serum should be used for testing. Should a delay

in testing occur, store the samples at 2 to 8°C. Samples can

be stored for up to 3 days at 2 to 8°C, provided they are not

contaminated. Do not use hemolyzed, icteric or highly

turbid sera. Turbid or particulate serum samples must be

clarified by centrifugation at 2000 rpm for 15 minutes. Use

the clear supernatant for testing.

Additional Material Required

Spectrophotometer with 505 to 578 nm wavelength

filters and cuvette mode, stopwatch, well-calibrated

micropipettes, disposable tips, isotonic saline, particulate

free distilled water, test tube rack, incubator/waterbath

set at 37°C, optically clean disposable cuvettes such as

Quantiamate semimicrocuvettes/glass cuvettes.

Note: Though any filter between the wavelengths 505 to

578 can be used, optimum results are obtained with a filter

with 546 nm wavelength.

Test Procedure

Bring reagents and specimen to room temperature before

use.

Assay Conditions

Wavelength 546 nm

Reaction temperature 37°C

Cuvette 1 cm path length

Method for Preparation of RF Calibration Curve

The QUANTIA-RF calibrator must be reconstituted

exactly with 1.0 mL of distilled water, wait for 10 minutes,

gently swirl the vial till the solution attains homogeneity.

Once reconstituted, it is ready to use for preparation of

RF calibration curve. The concentration (S) of RF in the

reconstituted calibrator is as mentioned.

Dilute the calibrator serially as mentioned below for

preparation of calibration curve.

Test tube No. 1 2 3 4 5

Calibrator dilution No. D1 D2 D3 D4 D5

Isotonic saline - 100 µL 100 µL 100 µL 100 µL

Calibrator

100 µL 100 µL 100 µL 100 µL 100 µL

Conc. of IgA in IU/mL 120 60 30 15 7.5

The above five dilutions of the calibrator including

the highest 120 IU/mL (D1) and lowest 7.5 IU/mL (D5)

concentrations of measuring range must be used for the

preparation of the calibration curve.

Test Procedure for Preparation of Calibration Curve

1. Zero the instrument with distilled water.

2. Pipette 400 µL of QUANTIA-RF activation buffer (R1)

and 100 µL of QUANTIA-RF latex reagent (R2) in the

measuring cuvette. Mix well and incubate for five

minutes at 37°C.

 or

 Pipette 500 µL of QUANTIA-RF working reagent in

the measuring cuvette. Mix well and incubate for five

minutes at 37°C.

3. Add 10 µL of calibrator (D1), mix gently and start the

stopwatch simultaneously.

4. Read absorbance (A1), exactly at 10 seconds, and

absorbance (A2) again at the end of exactly 4 minutes.

5. Repeat steps No. 2 to 4 for each diluted calibrator (D2

to D5) for preparing calibration curve.

6. Calculate DA (A2-A1) for each calibrator (D1 to D5).

Plot a graph of DA versus concentration of RF on the

graph paper provided with the kit.

“The calibration curve” so obtained is valid only for the

same lot of QUANTIA-RF reagents.

Test Procedure for Specimen

For determination of RF concentration in the test

specimen:

1. Follow steps 2 to 4 as mentioned in the above procedure

for calibration curve using the test specimen in place

of the calibrator.

2. Calculate ∆A (A2-A1) for the test specimen.

Validation Criteria

If the ∆A of the test specimen is less than ∆A obtained

for the standard of highest concentration (D1) then

the concentration of IgA in the test specimen can be

determined directly by interpolating ∆A of the test

specimen from the calibration curve.

If the ∆A of the diluted test specimen is higher than

∆A of standard with highest concentration (D1), then the

720 Concise Book of Medical Laboratory Technology: Methods and Interpretations test has to be rerun by carrying out further dilution of test

specimen such as 1:10, 1:20, etc. till the DA of the diluted

test specimen is less than ∆A of ∆1. Then proceed for calculations.