OnSite RF Rapid Test An Accurate, Simple,

Point of Care Test

Content

Rheumatoid Factor

Detection Methods

OnSite RF Detection

Content

Rheumatoid Factor

Rheumatoid Factor

• Human auto-antibodies that bind to the Fc moiety of immunoglobulins.

• The majority of RF are IgM subtypes, but it can be of IgG, IgA, IgE, or IgD subtypes.

IgA RF is seen in ~90% of RA patients, IgG RF is seen in ~70% of RA patients Detection of all Igs of RFs is required.

IgM RF IgG RF IgA RF

What RF Does • Antibodies are produced by the immune system to help

destroy and eliminate invading bacteria and viruses that can cause disease.

• The RF antibody can attach to normal body tissue, resulting in damage.

Elevated RF Value • Rheumatoid arthritis (60-70%)• Sjögren's syndrome (85-95%)• Felty's syndrome (>95%)• Systemic sclerosis (~30%)• SLE (~25-35%)• 5% of healthy persons and might be in 20% of the population over the

age of 65 years. • Less than one-third of children who have juvenile idiopathic arthritis

have a high RF level. • A number of connect tissue and inflammatory diseases

– Infectious mononucleosis Infective endocarditis– Dermatomyositis Hepatitis– Malignancy HIV, Syphilis – Leukaemia Tuberculosis– Sarcoidosis Influenza

Clinical Value of RF Detection

• Is the most often used to help diagnose rheumatoid arthritis or Sjogren syndrome.

• Prognosis of rheumatoid arthritis. o The higher the level in rheumatoid disease the worse

the joint destruction and the greater the chance of systemic involvement.

Content

Detection Methods

RF Value Varies • RF vary with subtypes, test methods and laboratories.

– IgM RF was reported to be > 6-25 IU/ml,– IgA RF to be > 4 IU/ml, and – IgG RF to be > 20-60 IU/ml

• Over 70% of patients with rheumatoid arthritis and over 60% of patients with Sjogren syndrome have RF levels ≥20 IU/ml

What Affects RF Value• Blood that is very high in fats.• Age. some people older than age 65 have a high RF level.• Having had multiple vaccinations or blood transfusions.• Extremely high RF levels (> 5,000 IU/ml) in serum may result in an

antigen excess condition producing a low RF value, If the patient’s clinical condition does not correlated with the RF result, dilute the specimen and repeat the test.

• Up to 20% RF patients do not have RF or the levels of RF is below the limit of detection. Repeating test can help

• A decrease in serum RF levels has been reported in associate with the response to latest treatment., such as in Ratuximab treatment.

RF Detection Methods

• Agglutination Test • Nephelometry Test• ELISA Test• Rapid Test

Agglutination Tests

• Series sample dilution prior to testing • Detects IgM RF • Shaking slide could lead to reagent

spillage • The agglutination particles are difficult

to observe, leading human error• Time consuming if testing large number

specimens. • Result is reported as titer ; > 1:80 titer

is positive

Nephelometry Test• Need clinical analyzer & trained personnel

to perform the test– Require constant electricity

• A quantitative test method for detection of IgM or IgG

• 40-60 IU/ml is the cut off • Prone to false positive, as only one

antibody is used. • EDTA anticoagulant leads to 59% increase

of value.

RF ELISA Test

• Require ELISA reader and skillful technician to perform the test

• Quantitative detection of RF individual subtypes

• Reported as IU /ml• Not popular

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OnSite RF Detection

OnSite RF Rapid Test

Kit Components:• 30 Individual Pouchs

– Test device – Dessicant

• 30 Capillary Tubes (5 μL) • 1 Sample Diluent • 1 Package Insert

OnSite RF Rapid Test Principle

Antibody Based Sandwich Lateral Flow Immunoassay to detect all isotypes of RF : IgM, IgG, IgA, IgE, and IgD

OnSite RF Rapid Test: Simple Procedure

Negative, no RF

Positive, RF>= 8IU/ml

Invalid, repeat

Step 1: Add 5 ul specimenSerum, plasmawhile blood

Step 2: Add 2 drops of sample diluent

Step 3: Wait 10 minutes to read results

OnSite RF Rapid Test: Sensitivity 8 IU/ml Matrix were spiked with 9 groups of RF to the WHO reference 2nd revision concentrations of 0, 1, 2, 4, 6, 8, 10, 15, and 20 IU/ml. The specimens were run on the OnSite RF Rapid. Results are presented in Table below:

Analytic Sensitivity is 8 IU/ml

RF IU/ml

# positive

# negative

Positive rate (%)

0 0 0 0%

1 0 20 0%

2 0 20 0%

4 6 14 30%

6 13 7 65%

8 19 1 95%

10 20 0 100%

15 20 0 100%

20 20 0 100%

0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 210

10

20

30

40

50

60

70

80

90

100

110

RF (IU/ml)

negative positive1

10

100

1000

10000

Test results OnSite RF Rapid TEst

OnSite Rapid Test: No Hook Effect

LOD (8 IU/ml)

0 8,400 (IU/ml)

OnSite Rapid Test Specificity The specificity was determined from studies with specimens from various disease stages. The results are presented in the Table below. No cross-reaction was observed, except the HAMA specimens which is not uncommon.

Specimens Size OnSite RF Rapid Test

Specimens Size OnSite RF Rapid Test

ANA positive 6 Negative Dengue Ab positive 10 Negative

HAMA positive 6 4 Negative H.Pylori Ab positive 10 Negative

HAV Ab positive 10 Negative Malaria positive 10 Negative

HBsAg positive 10 Negative TB Ab positive 10 Negative

HCV Ab positive 10 Negative Typhi Ab positive 10 Negative

OnSite Rapid Test Accuracy: To Nephelometry Test

OnSite RF Rapid Test

Nepholometry Positive Negative Total

RF > 20 IU/ml 40 3 43

8 IU/ml > RF > 20 IU/mL 43 2 45RF < 8 IU/mL 0 100 100

Total 83 105 188

188 specimens were tested.

For RF > 8 IU/ml, the detection agreement is 94% - RF > 20 IU/ml, the detection agreement is 93% - 8 IU/ml > RF > 20 IU/ml , the detection agreement is 95.5%For RF < 8 IU/ml , the detection agreement is 100% PPV: 100%; NPV: 95%

OnSite Rapid Test Accuracy: To Latex Agglutination Test

60 clinical specimens were Tested. The OnSite RF Rapid Test detected all of the specimens accurately. The latex agglutination test detected 95% of positive specimens and 100% of negative specimens

Specimen Number Latex Agglutination OnSite RF Rapid Test

RF ≥ 20 IU/ml 22 95.5% Positive 100% Positive

8 IU/ml ≤ RF < 20 IU/ml

18 100% Positive 100% Positive

RF < 8 IU/ml 20 100% Negative 100% Negative

OnSite RF Rapid Test is more accurate than Agglutination Test

OnSite Rapid Test Interference The interference of chemicals commonly found in OTC, prescription, blood components was studied by spiking these substances into 3 levels of RF specimens. The results shows that there was no interference observed.

OnSite RF Rapid Test Result SpecimensChemical Spiked

Zero RF weak positive

RF strong positive

Control - + ++

Albuman 50 g/l - + ++

Bilirubin 20 mg/dl - + ++

Creatine 442 umol/l - + ++

EDTA 3.4 umol/l - + ++

Glocuse 55 mmol/l - + ++

Heparin 3,000 U/l - + ++

Human IgG 1,000 mg/dl - + ++

Salicylic acid 4.34 mmol/l - + ++

Sodium citrate3.8% - + ++

OnSite RF Rapid Test on General Population

Specimen Type Frozen specimens Fresh specimens Sample Size 100 100

Positivity Rate 3% 5%

5% positivity rate to the general population by OnSite RF Rapid Test is in agreement with the normal value of 5%

Lots Limit of Detection (WHO standard)

300 random clinical specimens

60 samples from RF labin India

F0322K2 8IU/ml 5.67% positive <8IU/ml: all negative>20IU/ml: 95% Positive

F0404K5 8IU/ml 5.67% positive <8IU/ml: all negative>20IU/ml: 95% Positive

F0410K1 8IU/ml 6.98% positive <8IU/ml: all negative>20IU/ml: 95% Positive

Inter-lot variation

0% variation 1.3% variation 0% variation

OnSite Rapid Test: High PrecisionThree lots of products had consistent results to variant test samples.

Calibrated against the World Health Organization 2nd revision of Rheumatoid Arthritis Serum, Human reference reagent

OnSite RF Rapid Test Traceability

The OnSite Method Vs. AlternativesOnSite RF Rapid Test Latex Agglutination Nephelometry Test ELISA

RF subtypes All subtypes IgM IgG, IgM Individual subtype

Pre-preparation No Dilute specimens, prone to mistake

Not compatible with EDTA anticoagulant

No

Test procedure Simple require rotation Load calibrator Complicated , need

Time result 5-10 minutes 2 minutes 3 minutes 1-2 hours

Result reading Visual, easy Could be difficult Need machine Need ELISA reader

Whole blood Whole blood No No No

Positive result T & C line (> 8 IU/ml) > 1:80 titer > 40- 60 IU/ml > 40- 60 IU/ml

Storage 2-30⁰C 2-8⁰C 2-8⁰C 2-8⁰C

Shelf life 24 months 12 months 12 months 12 months

Small lab Well suited Suited No common Time consuming

OnSite RF Rapid Test is an accurate , simple, POC test

What Else About RF

The most common cause of false positive immunoassays

RF causing artificial binding of Ab-RF-Ab

Normal Ab-Ag-Ab binding

RF

Conclusion

• The OnSite RF Rapid Test is an accurate & simple test for quick screening of RF in aid of the diagnosis of RA and Sjogren syndrome

• It can be also used as a quick method to verify if the positive immunodiagnostic result is truely positive or caused by RF factor.

Effective RF Detection Algorism Screening by RF Rapid Test

Positive > 8

IU/ml

Nephelometry test for quantitative value

Prognosis

ELISA for RF quantitative value

Prognosis

Next RA tests

ANA

Anti-CCP

CRP

ESR, or sediment rate

Negative <

8 IU/ml

Follow up

The OnSite RF Rapid Test Can Be Used • The first line screening test to replace RF agglutination test • Can be used at the Rheumatologist office.• The interior / village market as well as to those customers where

work load is less and do not have biochemistry Analyzers. • Verifying of the board line result by nepholometry assay.• Field screening for mass population under Govt. project.• To eliminate the doubt on positive result in immunodiagnostic assay.

For further information about OnSite RF Rapid Test

Contact Info.:10110 Mesa Rim Rd. San Diego, CA 92121 USAEmail Address: tech@ctkbiotech.com Telephone: 1 (858) 457-8698Fax: 1 (858) 535-1739

Thank You !

QUALITY . INNOVATION . SIMPLICITY

Immunoturbidimetric assay

• It is quantitative measurement• The test is adoptable in Biochemistry Analysers• Cost per test is low• Calibration is stable• Little human error as seen in the latex agglutination assay • Most of the labs are equipped with Biochemistry Analysers• The instrument landing cost is 1,000USD and cost per test

0.5 USD with quantitative result.

Reference (please leave this in your edit revison, not published revision

Wilson D; Rheumatoid factors in patients with rheumatoid arthritis. Can Fam Physician. 2006 Feb;52:180-1.Rheumatoid arthritis: the management of rheumatoid arthritis in adults, NICE Clinical Guideline (February 2009) Pincus T; Advantages and limitations of quantitative measures to assess rheumatoid arthritis - joint counts, radiographs, laboratory tests, and patient. Bull Hosp Jt Dis. 2006;64(1-2):32-9.Shin YS, Choi JH, Nahm DH, et al; Rheumatoid factor is a marker of disease severity in Korean rheumatoid arthritis. Yonsei Med J. 2005 Aug 31;46(4):464-70.Nishimura K, Sugiyama D, Kogata Y, et al; Meta-analysis: diagnostic accuracy of anti-cyclic citrullinated peptide antibody and rheumatoid factor for rheumatoid arthritis. Ann Intern Med. 2007 Jun 5;146(11):797-808.Longmore M, Wilkinson IB and Rajagopalan SR (2004): Oxford Handbook of Clinical Medicine, 6th ed, OUPChen HA, Lin KC, Chen CH, et al; The effect of etanercept on anti-cyclic citrullinated peptide antibodies and rheumatoid factor in patients with rheumatoid arthritis. Ann Rheum Dis. 2006 Jan;65(1):35-9. Epub 2005 Jun 23.De Rycke L, Verhelst X, Kruithof E, et al; Rheumatoid factor, but not anti-cyclic citrullinated peptide antibodies, is modulated by infliximab treatment in rheumatoid arthritis. Ann Rheum Dis. 2005 Feb;64(2):299-302. Epub 2004 May 27.Vittecoq O, Pouplin S, Krzanowska K, et al; Rheumatoid factor is the strongest predictor of radiological progression of rheumatoid arthritis in a three-year prospective study in community-recruited patients. Rheumatology (Oxford). 2003 Aug;42(8):939-46. Epub 2003 Apr 16.