17 Reading, Grading, and Recording Reactions
Principle
Hemolysis and agglutination are both visible endpoints to an antigen-antibody reaction. Hemolysis is only considered a positive reaction when using fresh serum samples and polyspecific antihuman globulin or anti-C3d. Complement cannot be activated in EDTA plasma samples as the calcium is chelated. Standardization of the reading, grading, and recording of the agglutination reactions is critical to the uniformity and reproducibility of test results. Use of an optical aid, such as an agglutination viewer with mirror enhances the consistency. A microscope has limited applications such as the confirmation of the presence of mixed-field reactions. Routine microscopic examination is not required.
Related Policies
This procedure applies to all testing that requires the reading, grading, and recording of agglutination test results.
Procedure
- Centrifuge the test tubes for the designated amount of time and speed.
- Inspect the serum/plasma/reagent surrounding the cell button for hemolysis. Record hemolysis if present as “H”.
- Gently shake the tube to suspend the cell button from the bottom of the tube. The tilt technique uses the meniscus to gently dislodge the red cell button from the wall of the tube. Observe the way the cells leave the red cell button.
- Stop shaking the tube when the cell button is free (dislodged from the bottom of the tube).
- Observe the suspended cell button. Negative reactions appear as a smooth homogeneous, opaque suspension of cells. Positive reactions will appear as “chips” or “chunks” of agglutinates in a clear or cloudy background.
- Grade results according to interpretation of agglutination reactions.
- If indicated by the method, observe “questionable” agglutination results microscopically, using the inverted microscope.
- Read, grade and immediately record reactions while tubes are in your hand.
Interpretation
- Agglutination or hemolysis of the red blood cells is a positive test result.
- No agglutination or hemolysis of the red blood cells is a negative test result.
- The following may be used as a guideline for interpretation of positive result recording:
| Reaction Strength/Score | Appearance |
|---|---|
| 4+ (12) | A single solid agglutinate. No free red cells. |
| 3+ (10) | A few large agglutinates. Clear background. |
| 2+ (8) | Many medium-sized agglutinates. Clear background. |
| 1+ (5) | Many small agglutinates in a cloudy background. |
| w (2) | Barely visible agglutination in a cloudy background |
| 0 (0) | No agglutinates with an even red blood cell suspension, and no hemolysis seen. |
| m | Appears macroscopically negative. Agglutinates of 6-8 red blood cells in most microscopic fields. |
| mf | Mixed-field agglutination noted either macroscopically as agglutinates in a suspension of opaque red blood cells or microscopically as a few well-defined agglutinates with many free cells. |
| R | Rouleaux. Appears as cells stacked like coins microscopically. |
| H | Hemolysis* see note #5. |
Notes
- Shaking the tube too hard or too lightly may result in false results.
- Shaking the tubes after the cell button is suspended may reduce the reaction strength.
- Microscopic observation (if indicated by the method) of agglutination tests may result in over-reading.
- Microscopic (if indicated by the method) mixed-field reactions may be a result of bone marrow/stem cell transplantation, transfused neonates or trauma patients (non-ABO specific red cells).
- Hemolysis may be a result of activation of the complement cascade by an antibody/antigen reaction. Antigen/Antibody activated hemolysis cannot occur with EDTA plasma samples as the C1qrs complex requires Ca++ and Ca++ is chelated by EDTA. Complement activation can occur in fresh serum samples and observed when using polyspecific antihuman globulin or anti-C3d.