7-6 SEROLOGY

LEARNING OBJECTIVE: Recall principles and procedures for the Rapid Plasma Reagin (RPR) Card Test and the Monosticon DRI-DOT® Slide Test.

Serology consists of procedures by which antigens and reacting serum globulin antibodies may be measured qualitatively and quantitatively. Serologic tests have been devised to detect either antigens present or antibodies produced in a number of conditions. Most tests are based on agglutination reactions between an antigen and a specific antibody.

An antigen is a substance that, when introduced into an individual who does not already possess that substance, may stimulate the individual’s cells to produce specific antibodies that react to this substance in a detectable way. The five basic characteristics of an antigen are that it must be foreign to the body, it must possess a high molecular weight, it must be structurally stable, it must be complex, and it must have a high specificity to stimulate tissues to produce a defensive protein substance called an antibody.

Antibodies are the specific defensive proteins produced when an antigen stimulates individual cells. Antibodies are produced by the host in response to the presence of an antigen and are capable of reacting with antigens in some detectable way.

The antigen-antibody reaction takes place when a reaction occurs between specific antibodies in the plasma and the antigen present on cell surfaces.

Principles and procedures of two serologic tests, the rapid plasma reagin (RPR) card test and the Monosticon DRI-DOT® Slide Test are covered in the following sections.

RAPID PLASMA REAGIN (RPR) CARD TEST

The RPR Card test is a sensitive, easily performed screening test for syphilis. The test is performed on unheated plasma or serum. Everything needed for the test is in a kit that is available commercially. This test kit is very useful aboard ship and at small stations.

Principle of the RPR Card Test

In the RPR Card test method of syphilis detection, a specific antigen (carbon-particle cardiolipin) detects “reagin,” a substance present in the serum of persons who are infected with syphilis. Specimens that contain reagin cause formation ofparticles (called flocculation) or coagulation of the carbon particles to occur on the RPR Card antigen. Reactive specimens appear as black clumps against a white background. Nonreactive specimens appear as an even, light-gray color.

Materials Required for RPR Test

To perform an RPR Card test, the following materials are required:

NOTE

Use clear, unhemolyzed serum that has been separated from the blood cells as soon after collection as possible.

—RPR Card antigen suspension

—Plastic dispensing bottle

—20-gauge, galvanized needle, blunt cut

—Test cards

—Pipette/stirrers, 50 microliter (1ul)

· RPR Card Test Control Cards (each consisting of three labeled test areas containing lyophilized (meaning a stabilized preparation of a biological substance, such as blood, that has been frozen rapidly and then dehydrated under a high vacuum) control specimens with designated patterns of reactivity: Reactive, Reactive­Minimal-to-Moderate, and Nonreactive.)

NOTE

RPR Card Test Antigen and Control Cards must be stored at 4°C when not in use. Both items are stable until the expiration date. Store “in use” antigen suspension in the dispensing bottle at 4°C. The antigen suspension is stable for 3 months or until the expiration date, whichever occurs first.

Preliminary Preparations for RPR Test

The following preliminary preparations must be performed before RPR testing can begin:

  1. Remove the antigen suspension vial and one control card envelope from the refrigerator. Allow the items to warm to room temperature.
  2. Resuspend the contents of the vial by vigorously shaking the antigen vial.
  3. Snap the neck of the vial.
  4. Attach the needle (provided in the kit) to a 1 ml tuberculin syringe. Slowly draw up into the syringe from the vial approximately 1 ml of the antigen suspension.
  5. Hold the syringe perpendicular to the surface and count the number of drops dispensed from a 0.5 ml volume. Allow the drops to fall into the antigen vial.

NOTE

The needle is accurate if 30 drops, plus or minus 1 drop, are dispensed from the 0.5 ml volume.

  1. Slowly expel the remainder of the antigen solution in the syringe back into the antigen vial.
  2. Remove the needle from the syringe. Place the needle on the tapered fitting of the plastic dispensing bottle (provided in the kit).
  3. Slowly withdraw all the contents of the antigen vial by collapsing the dispensing bottle and using it as a suction device.
  4. Allow the rotator to warm up for 5 to 10 minutes; adjust to 100 rpm.

RPR Test Procedure

To detect syphilis using the RPR Card test, follow the steps below:

  1. Open the foil package and remove the control card.
  2. Use a pipette/stirrer to reconstitute each control card circle with 0.5 ml of distilled water.
  3. Mix solution in control card circle with pipette/stirrer until the dehydrated control specimen is dissolved. Spread specimen over entire area of circle. Use a separate pipette/stirrer for each circle.
  4. Draw the patient’s sample by holding the pipette/stirrer between the thumb and forefinger near the stirring or sealed end and squeeze. Do not release pressure until the open end is below the surface of the specimen, then release pressure to draw up the sample.
  5. Hold the pipette/stirrer in a vertical position, directly over the card test area where the specimen is to be delivered; squeeze pipette/stirrer, allowing 1 drop to fall onto the test area.
  6. Invert the pipette/stirrer and, with the sealed end, spread specimen within the circle. Discard the pipette/stirrer when done.
  7. Continue the steps above until one or two test cards are filled with patient’s samples.
  8. Gently shake the antigen dispensing bottle before use. Hold the bottle in the vertical position and dispense several drops into the dispensing bottle cap to ensure the needle passage is clear. Allow 1 “free-falling” drop to fall onto each test area. Do not stir; the mixing of antigen suspension and specimen is accomplished by rotation.
  9. Put the card(s) on the rotator and cover with the humidifying cover. Rotate cards for 8 minutes at 100 rpm. To help differentiate nonreactive from reactive results, you should briefly rotate and tilt the card by hand (3 or 4 back-and-forth motions).
  10. Immediately read the card macroscopically (with the unaided eye) in the “wet” state under a high-intensity lamp.
  11. Compare the patient’s tests to the controls for correct interpretations. The reactive control should show small to large clumps. The nonreactive control should show no clumping or very slight roughness. The reactive-minimal­to-moderate control should show slight but definite clumping.
  12. Report the test as:

NOTE

The RPR Card test is used as a screen for syphilis. If a patient’s RPR is reactive, the patient should be sent to a laboratory to have a FTA-ABS (Fluorescent Treponemal Antibody Absorption Test) performed. The FTA-ABS, a more precise test, is used to confirm primary, secondary, and late syphilis.

MONOSTICON DRI-DOT SLIDE TEST

Mononucleosis imitates many diseases so well that diagnosis is confirmed only by selective serologic testing. The Monosticon DRI-DOT Slide Test is an accurate, 2-minute disposable test designed to detect the presence of infectious mononucleosis antibodies in serum, plasma, or whole blood.

Principle of the Monosticon DRI-DOT Slide Test

The Monosticon DRI-DOT Slide Test consists of specially prepared, stable sheep and/or horse erythrocyte antigen (dyed) and guinea pig antigen on a disposable slide. When serum, plasma, or whole blood is mixed with these antigens on the slide, the test result for infectious mononucleosis will be positive or negative. A positive result is indicated by agglutination and a negative result is indicated by no agglutination.

Materials Required for Monosticon DRI-DOT Slide Test

To perform the Monosticon DRI-DOT Slide Test, the following materials are required:

  1. Serum or plasma specimen
  2. Monosticon DRI-DOT Test kit, which consists of:
—Monosticon DRI-DOT Test slides
—Positive I.M. (infectious mononucleosis) serum control
—Negative I.M. serum control —Dropper bottle
—Dispenstirs® (designed to deliver a 0.03 ml drop)
  1. Distilled water
  2. Centrifuge
  3. DRI-DOT slide holder (available commercially, but not necessary to perform test)

Controls for Monosticon DRI-DOT Slide Test

Both a positive and negative control are included in each kit to check the effectiveness of the reagents. The positive I.M. serum control (human) is a dilution of human sera (sing. serum) containing the specific heterophile antibody of infectious mononucleosis. The negative I.M. serum control (human) is a dilution of human sera containing no detectable antibody to infectious mononucleosis. Both controls have been dried and placed in a vial with color-coded cap and label. Since both controls are of human origin, they are potentially infectious and must be handled with care.

Both controls (positive and negative) should be tested before performing test with serum, plasma, or whole blood. Controls are prepared in the same manner as serum and plasma test described in the next section, but instead of adding serum or plasma to the slide, the control is added. Before each control is used, it must be reconstituted with 0.5 ml ofdistilled water. Ifresults of the control tests are not as expected, do not use the test kit. 

Monosticon DRI-DOT Slide Test Procedure

To detect mononucleosis using the Monosticon DRI-DOT Slide Test, follow the steps below.

  1. Centrifuge the blood specimen for 10 minutes to obtain the plasma or serum to be tested.
  2. Fill the dropper bottle with distilled water.
  3. Remove the disposable slide by tearing the envelope where indicated. (Remove only enough slides to perform the tests at hand.)
  4. Set the slide in a holder or on a flat surface.
  5. Place one drop of water from the dropper bottle next to but not on the blue dot within the circle on the slide.
  6. Use a Dispenstir to squeeze the closed end between thumb and forefinger, and place the open end into the plasma or serum to be tested. Release pressure to draw up the specimen into the Dispenstir.
  7. Hold the Dispenstir perpendicularly over the buff-colored dot (guinea pig antigen) within the circle of the slide. Place one drop of specimen onto the dot.
  8. Use the flared end of the Dispenstir to mix the water, specimen, and the guinea pig antigen (bluff-colored dot) thoroughly.
  9. Blend this mixture thoroughly with the blue dot (horse/sheep antigen).
  10. Rock the slide (or slide holder) back and forth gently in a figure-8 motion for 2 minutes so that the liquid slowly flows over the entire area within the circle.
  11. After 2 minutes, read the results under a strong, glaring light.
  12. Report test as

See figure 7-21 for an illustration of positive and negative test results.

Figure 7-21.—Illustration of positive and negative Monosticon DRI-DOT Slide Test Results.

NOTE

A positive test result usually occurs between the fourth day and the twenty-first day of illness, and may persist for several months.