A blood bank scientist must exercise caution and careful consideration of probabilities when evaluating a suspected antibody, particularly one with clinical significance. Each phase of reactivity, as previously discussed, along with the controlled conditions of each stage—including temperature, centrifugation, and washing—is crucial for ensuring optimal antibody/antigen interaction. It's also important to acknowledge that each testing method and phase comes with its limitations. A negative reaction does not necessarily mean the absence of the antibody in the patient.
Antibodies have optimum temperatures for reactivity. Reaction readings can be made at different phases—after the immediate spin, after incubation at 37°C, and after the addition of antihuman globulin (AHG) and centrifugation. Reactivity in a certain phase will help to determine whether the antibody is cold reacting (IgM) or warm reacting (IgG). It will also help to distinguish between antibodies that are clinically significant and not significant. Clinically significant antibodies that are capable of causing acute and delayed hemolytic transfusion reactions (HTR) or hemolytic disease of the fetus and newborn (HDFN) are usually IgG and react best in the AHG phase. See Table 2 for general "rule of thumb" characteristics of antibody reactions.
Three commonly used phases describe the likely presence or absence of an antibody. The test tube method is a popular technique for conducting these evaluative phases. Each phase is designed to replicate various temperature environments within the human body. Clinically significant antibodies, for instance, generally show reactivity in the warmer phases that are closer to body temperature.
- Immediate spin: Antibodies reacting in this phase tend to be cold reactive (cold referring to temperatures below the standard human body temperature). Cold reactive antibodies are usually IgM class and not clinically significant (with the exception of the A and B antibodies).
- 37°C: Antibodies reacting in this phase typically include strong IgM or IgG antibodies. Following incubation, the tubes are examined for the presence of hemolysis. During this phase, if an antibody that can recruit the complement system is present, it will bind to the target antigen on the red blood cells. This binding triggers a cascade of reactions involving the complement proteins, leading to the potential for cell lysis or hemolysis. Observing hemolysis at this stage indicates that complement has successfully bound during the incubation, highlighting the presence of antibodies that can activate the complement pathway, which is an essential consideration for ensuring transfusion compatibility and patient safety. Note: If EDTA plasma samples are used for testing, the complement cascade has been halted. Magnesium and calcium ions are not available for complement to be activated.
- AHG: An antibody response in the AHG phase signifies clinical importance. This antibody, typically IgG, can sensitize at the in vivo temperature of the human body (warm temperatures), indicating its relevance to patient health. Antibodies reacting in this phase are considered warm reactive antibodies.
Table 2. Rule of Thumb for Antibody Reactions. | Immediate Spin | 37°C Incubation | Antihuman Globulin (AHG) |
| Antibody | Typically IgM "Cold reactive antibodies" | Strong IgM or IgG Examine for hemolysis | Typically IgG "Warm reactive antibodies" |
| Example antibodies that react | Lewis (anti-Lea)
P1
M
N | Rh (e.g., anti-D, anti-E,
anti-c) | Duffy
Kidd
Kell
S and s
Rh (e.g., anti-D, anti-E,
anti-c) |
| Clinical significance | Usually not clinically significant (except A and B) | Complement activating antibodies Clinically significant | Clinically significant Capable of causing HTR and HDFN |
