CHAPTER 12: Rapid Plasma Reagin (RPR) Carbon Agglutination Test syphilis

Treponema pallidum

what is the causative agent of syphilis

syphilis

sexually transmitted disease sometimes called as “the great imitator”

• symptoms include painless, firm, round, and small sore on mouth, anus and genitals

• some are asymptomatic for years but can have complications if untreated

Chancre

Appears on the primary stage of syphilis

Condylomata lata

appears on the 2nd stage of syphilis

Gummas

appear on 3rd stage of syphilis

nontreponemal/reagin antibodies, antibodies specific for T. pallidum

2 types of antibodies produced in response to T. pallidum infection

nontreponemal or reagin antibodies

antibodies produced by the infected individual directed against his own cell components

• also present in infectious disesases such as chickenpox, hepatitis, infectious mononucleosis, leprosy, tuberculosis, malaria, and etc

Rapid plasma reagin test

one of the several test used in the diagnosis of syphilis

• high sensitivity and stability

• mostly widely performed method for the detection of nontreponemal antibodies

• nontreponemal flocculation card test for syphilis that detects reagin, an antibody formed against cardiolipin during the course of infection

darkfield microscopy

• direct method

• labeled as test of choice

Fluorescence treponemal antibody adsorption/FTA-ABS test

most specific and confirmatory test

VDRL

earliest syphilis test, similar to RPR but has less stability and less sensitivity of RPR

Flocculation

_ occurs with coagglutination of the carbon particles, resulting in black clumps against the white background of the plastic-coated card.

antigen lot number, expiry, and transfer dates

(reagent prep) Label the dispensing bottle with ?

3 months

(reagent prep) stability is about _ or until expiry date, which ever comes first.

Carbon antigen, Controls, Disposable slides, Disposable dispensing pipettes, Mixing sticks, Rubber teats, Stopwatch, High intensity light source, Pipettes, NSS, Test tubes, Mechanical rotator, Needle dropper

Materials used in RPR experiment

plasma/serum

specimen used

• collected through venipuncture using aseptic technique

True

True or false: Heat inactivation is unnecessary

room temperature

In RPR Qualitative Test in Microtiter Plates, Controls, samples and reagents should be stored at what temperature before use?

1 drop

Using a flat-bottomed microtiter plate, place 50ul of the patient’s sample and _ drop of the control into separate circles on the card.

one

Add how many drop/s of free falling antigen to each test circle?

100 rpm, 8 minutes

Rotate the cards at _ rpm for how long?.

macroscopically, light box, white surface

Read _ , either over a _ or under a high intensity incandescent lamp over a _ surface

RPR Qualitative Test in Microtiter Plates

What test is this?

1. Controls, samples and reagents should be stored at room temperature before use.

2. Using a flat-bottomed microtiter plate, place 50ul of the patient’s sample and 1 drop of the control into separate circles on the card.

3. Resuspend the antigen gently.

4. Add one drop of free falling antigen to each test circle.

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 8 minutes.

7. Read macroscopically, either over a light box or under a high intensity incandescent lamp over a white surface.

Semi-Quantitative Test

What test is this?

1. Add 50ul of saline to circles 2, 3, 4, and 5 with the use of a semi-automatic pipette. Do not

spread the saline.

2. On circles 1 and 2, add 50ul of patient’s sample.

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 50ul from circle 2 to the saline in circle 3.

5. Perform serial dilutions in the same manner until the last circle, discarding 50ul 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 next sample until reaching circle 1.

7. Proceed as qualitative test from step 3.

t

t or f: Reactive sera may be tittered

R, reactive

Write Reading & report if observed agglutination: Medium and Large Clumps

W, weakly reactive

Write Reading & report if observed agglutination: Small Clumps

N, non-reactive

Write Reading & report if observed agglutination: No Clumping or Very Slight ‘Roughness’

9% saline

To titer, make serial two-fold dilutions using _ as described in Quantitative Procedure earlier on

serum titer

is defined as the highest dilution showing a positive result.