Serology: RA latex test kit --rapid agglutination --semi quantitative for rheumatoid factors --principle: Singer and Plotz

( 1956:1958) described a method of detecting Rheumatoid factor using a suspension of fine plastic granules coated with human gamma globulins which were agglutinated in the presence of rheumatoid factor. The RA latex rgt is a sensitive, standardized preparation of this type, made with a purified human IgG fraction and selected polysterene latex. The presence or absence of a visible agglutination indicates the presence or absence of RF in the samples tested. --composition: Std. kit contents: 1. Latex rgt: contains < 0.1% Azide 2. Positive control: human (+) RA serum with an activity >25IU/ml. 3. Negative control 4. 10x concentrated glycine buffer: add one part to nine parts distilled water before use. On dilution the diluent has a pH between 8.0 and 8.2 5. Single use pipette/stirrers, reusable agglutination slide 6. Kit insert --storage: upright at 2-8*C. do not freeze any rgts. --procedure: Qualitative method: 1. Allow each component to reach room temp. 2. Gently shake latex rgt. 3. Add 1 drop of latex rgt using the dropper provided (40ul) to each of the required circles of the agglutination slide. 4. Using the pipette stirrer, place a drop of undiluted serum onto a circle of a test slide. 5. Spread the rgt and serum sample over the entire area of the test circle using a separate stirrer for each sample. 6. Gently tilt the test slide backwards and forwards approx.. once every 2 seconds for two mins. Interpret results immediately after 2 mins. Extended incubation may lead to false results. Positive and negative controls should be included at regular intervals. Both are ready for use and do not require further dilution. 7. Rinse test slide with distilled water, dry and store in a sealed bag. Semi-quantitative determination 1. Can be performed in the same way as quali using serial dilutions of saline, phosphate buffered saline or glycine saline as follows: Dilutions 1/2 1/4 1/8 1/16 Sample serum 100ul Saline 100ul 100ul 100ul 100ul 100ul 100ul

Volume of 50ul samples 8x titre 8x2 I.U./ml. 16 Normal levels:-Adults <8 IU/ml.

50ul 8x4 32

50ul 8x8 64

100ul 50ul 8x16 128

Calculation of results: The titre is expressed as the reciprocal of the highest dilution showing macroscopic agglutination: e.g. if this occurs in dilution 3, the titre is 8 corresponding to a concentration 64 IU/ml. Interpretation of results: Presence of agglutination indicates a level of RF in the sample =/>8 IU/ml. The lack of agglutination indicates a level of RF in the sample of <8 IU/ml. Using a latex test system , positive results are not always found with every case of clinically defined rheumatoid arthritis. The number of positives reported using various types of latex reagent range from 70% to over 90%. False positives results also occur in various pathological conditions including LE, hepa, liver cirrhosis, lymphomas, scleroderma, and various other infections. The frequency of false positive results is not high even in these conditions but the possibility must be borne in mind when interpreting results. Performance characteristics: Analytical sensitivity: 8IU/ml (6-16IU/ml) Prozone effect: no prozone effect detected up to 800 IU/ml. Diagnostic sensitivity: 100% Diagnostic specificity: 98.9% Limitations of the method The incidence of false positive results is about 3-5% May give positive results: IM, hepa, syphilis, elderly people (30% aged 60 years +) Diagnosis should not be solely based on the results of latex mtd but also should be complemented with a Waaler Rose test along with clinical examination. NB: results obtained with a latex method do not compare with those obtained with Waaler Rose test. Differences in the results between mtds do not reflect differences in the ability to detect RF. Hemoglobin (10g/L), bilirubin (20 mg/dl), and lipemia (<10 g/l, do not interfere. Other substances may interfere.

C-REACTIVE PROTEIN (CRP) Principle: The CRP latex test is a rapid slide agglutination test for the qualitative and semi-quantitative detection of C-reactive protein in serum. The reagent containing particles coated with specific anti-human C-Reactive protein antibodies, agglutinates in the presence of CRP in the patients serum. Presentation: Contents 25 Tests 50 Tests 100 Tests 150 Tests CRP Latex 1 x 1.0 ml 1 x 2.0 ml 1 x 4.0 ml 2 x 3.0 ml Positive control 1 x 0.5 ml 1 x 0.5 ml 1 x 1.0 ml 1 x 1.5 ml Negative 1 x 0.5 ml 1 x .05 ml 1 x 1.0 ml 1 x 1.5 ml control Tests Cards 1 1 2 3 Reusable Pipette / 25 50 100 150 Stirrers Composition: CRP Latex

Positive Control Negative Control

Suspension of white latex particles coated with specific anti-human C-Reactive protein antibodies. The sensitivity has been adjusted to detect between 6mg/L and 250 mg/L of C-Reactive protein. Sodium azide 0.95 g/L Human serum Sodium azide 0.95 g/L Animal serum Sodium azide 0.95 g/L

Although all our components which have been derived from human origin have been tested and found to be negative for the presence of anti-HIV, anti-HCV as well as HbsAg, it is recommended that they be handled cautiously and treated potentially infectious. Storage: Store components at 2-8C. Cards and pipettes may be kept at room temperature. Samples: Serum stable for 48 hours at 2-8C. Samples should be free from contamination, hemolysis and lipemia. Additional Equipment: Mechanical rotator set at 100 rpm. Test Procedure:

1. 2. 3. 4. 5.

Bring the reagents and samples to room temperature. Place 50 l of the sample and 1 drop of the control into separate circles on the card. Resuspend the latex gently. Add one drop of the latex reagent to each circle next to the sample which is to be tested. Mix with the disposable pipette/stirrer and spread over the entire area enclosed by the ring. Use a new stirrer for each sample. 6. Rotate the cards at 100 rpm for 2 minutes. Quantitative Test: 1. Using a semi-automatic pipette, add 50 l of 9g/L saline to circles 2, 3, 4, and 5. Do not spread the saline. 2. Add 50l of patient sample to circles 1 & 2. 3. Mix the saline and sample in circle 2 by drawing the mixture up and down being careful to avoid the formation of any bubbles. 4. Transfer 50 l from circle 2 to the saline in circle 3. 5. Perform serial dilutions in the same manner until the last circle, discarding 50 l at the end. 6. Using the pipette/stirrer, spread the diluted samples over the entire area of each circle starting at circle 5 and working backwards to the neat sample in circle 1. 7. Proceed as a qualitative test from step 3. Quality Control: Each run of tests should be validated with a positive and negative control. Reading and Interpretation: Examine macroscopically for the presence or absence of clumps or agglutination within 1 minute of removing the card from the rotator. Positive results the presence of agglutination indicates a level of >6 mg/L Negative results no agglutination would indicate a level of CRP <6 mg/L Normal levels in adults - >6 mg/L. Positive sera may be titred. To titrate make serial two-fold dilutions in 9 g/L saline as indicated in the quantitative test procedure. For example: The serum titer is defined as the highest dilution showing macroscopic agglutination. The approximate CRP concentration in the sample may be obtained by multiplying the titer by the limit of sensitivity 6 mg/L. Dilution Neat 1:2 1:4 1:8 1:16 CRP mg/L 6 12 24 48 96

Note: CRP has been detected in serum obtained from apparently healthy adults and children. The reported mean value ranged from 0.1 mg/L in newborns to 0.5 g/L in male adults. The CRP level can increase significantly above the normal levels with the onset of substantial information inflammatory stimulus. Limitations of the Procedure: CRP levels in the range of 15 mg/L or above may cause false negative results due to prozone effects. A final diagnosis should not be made on the result of a single test but should be based on a correlation of test results with other clinical findings. Notes: 1. The sensitivity of the test may be reduced at low temperatures. The best results are obtained over 10C. 2. Delay in reading the results may result in over-estimation of the CRP level. ANTI STREPTOLYSIN O (ASO) Principle: The ASO-Latex test is a rapid slide agglutination test for the direct detection and semi-quantitation of anti-streptolysin (ASO). The antigen, a particulate latex suspension coated with streptolysin O, agglutinates in the presence of specific antibodies present in the sera of patients with Streptococcal hemolytic infection (group A and C). Presentation: Contents ASO Latex Positive control Negative control Tests Cards Reusable Pipette / Stirrers Composition: ASO Latex Positive Control Negative Control

25 Tests 1 x 1.0 ml 1 x 0.5 ml 1 x 0.5 ml 1 25

50 Tests 1 x 2.0 ml 1 x 0.5 ml 1 x .05 ml 1 50

100 Tests 1 x 4.0 ml 1 x 1.0 ml 1 x 1.0 ml 2 100

150 Tests 2 x 3.0 ml 1 x 1.5 ml 1 x 1.5 ml 3 150

Suspension of white latex particles coated with streptolysin O. Sodium azide 0.95 g/L Stabilized serum Sodium azide 0.95 g/L Animal serum Sodium azide 0.95 g/L

Although all our components which have been derived from human origin have been tested and found to be negative for the presence of anti-HIV, anti-HCV as well as HbsAg, it is recommended that they be handled cautiously and treated potentially infectious. Storage: Store components at 2-8C. Cards and pipettes may be kept at room temperature. Samples: Serum stable for 48 hours at 2-8C. Samples should be free from contamination, hemolysis and lipemia. Additional Equipment: Mechanical rotator set at 100 rpm. Diluent: normal saline for dilution of samples (9 g/L) Qualitative Test Procedure: 1. Bring the reagents and samples to room temperature. 2. Place 50 l of the sample and 1 drop of the control into separate circles on the card. 3. Resuspend the latex gently. 4. Add one drop of the latex reagent to each circle next to the sample which is to be tested. 5. Mix with the disposable pipette/stirrer and spread over the entire area enclosed by the ring. Use a new stirrer for each sample. 6. Rotate the cards at 100 rpm for 2 minutes. Quantitative Test: Quantitative estimation of ASO can be carried out by using 2 different procedures. Both procedures yield identical results. Procedure II should be used for higher dilution series. Procedure I: 1. Using a semi-automatic pipette, add 50 l of 9g/L saline to circles 2, 3, 4, and 5. Do not spread the saline. 2. Add 50 l of patient sample to circles 1 & 2. 3. Mix the saline and sample in circle 2 by drawing the mixture up and down being careful to avoid the formation of any bubbles. 4. Transfer 50 l from circle 2 to the saline in circle 3. 5. Perform serial dilutions in the same manner until the last circle discarding 50 l at the end. 6. Using the pipette/stirrer, spread the diluted samples over the entire area of each circle starting at circle 5 and working backwards to the neat sample in circle 3. 7. Proceed as a qualitative test from step 3. Dilution Neat ASO IU/ml 200

1:2 1:4 1:8 Procedure II: Dilution

400 800 1 600

Sample undiluted

Diluent

Latex reagent

NEAT 1+1 1+2 1+3 1+4 1+5 1+6 1+7

50 l 50 l 50 l 50 l 50 l 50 l 50 l 50 l

50 l 100 l 150 l 200 l 250 l 300 l 350 l

50 l 50 l 50 l 50 l 50 l 50 l 50 l 50 l

ASO in IU/mL (Undiluted sample conc.) 200 400 600 800 1 000 1 200 1 400 1 600

Quality control: Each run of tests should be validated with a positive and negative control.

Typhidot: Reagents/ procedure: 1. Diluent( 200ul)+ serum (10 ul) (20 mins) 2. Enzyme conjugate=100ul (20 mins) 3. Substrate=100ul (10 mins) 4. Stop solution=100 Spectro: 450nm