Chapter 09 Detection and Identification of Antibodies

• Detection of red blood cell antibodies is vital for pretransfusion compatibility testing.
• It is a key tool for investigating hemolytic transfusion reactions and immune hemolytic anemias.
• It is also important for detecting and monitoring patients at risk of delivering infants with hemolytic disease of the fetus and newborn (HDFN).
• Antibody detection targets "irregular" or "unexpected" antibodies, excluding ABO system antibodies.
• Immune alloantibodies are primary, produced from red blood cell stimulation via transfusion, transplantation, or pregnancy
• Naturally occurring antibodies form without red blood cell stimulation from environmental sources. Passive antibodies are transferred via blood components.
• Clinically significant antibodies decrease red blood cell survival with the target antigen; they are typically IgG and react at 37°C or in the antihuman globulin phase of the indirect antiglobulin test.
• An antibody identification panel determines the antibody specificity after detection.

Antibody Screen

• Only a small percentage of the population (0.2% to 2%) has detectable red blood cell antibodies, but careful screening is still required for certain populations.
• The AABB's Standards requires an antibody screen to detect clinically significant antibodies in both blood donors and intended recipients for pretransfusion compatibility testing.

Tube Method

• The traditional method, using an indirect antiglobulin test where red blood cell reagents, enhancement reagents, and antihuman globulin reagents are used to sensitize red blood cells with patient antibodies.
• A 37°C incubation phase is required for IgG antibodies to sensitize red blood cells, potentially enhanced by adding enhancement media.
• The tubes are centrifuged, and observed for hemolysis and agglutination. The supernatant is examined for pink or red discoloration. Agglutination is graded from 0 (no agglutination) to 4+ (one solid agglutinate).
• Tubes are washed with saline to remove unbound antibodies and centrifuged and examined for hemolysis and agglutination with Coombs' serum.

RBC Reagents

• Come from group O individuals who have been typed for common and significant red blood cell antigens. Group O cells are used to prevent anti-A and anti-B interference.
• The RBCs are at a concentration between 2% and 5% in a preservative diluent, which maintains integrity and prevents hemolysis
• Packaged in sets of two or three cell suspensions, each having a unique combination of antigens, with at least one cell positive for antigens such as D, C, c, E, e, K, k, Fya, Fyb, Jka, Jkb, Lea, Leb, P1, M, N, S, and s.
• Ideally, there will be homozygous expression of many of the antigens within the screen cell set. Antibodies that react more strongly with cells having homozygous antigen expression are said to show dosage.
• When testing donors, a pooled screening reagent of red blood cells from at least two individuals can be used with careful observation for mixed-field agglutination. Commercially prepared cells are superior to crossmatching alone.

Enhancement reagents

• Added to increase the sensitivity of the test system or shorten the incubation time.

22% Albumin

• Reduces the zeta potential and dispersing the electrostatic charges, increasing the chances of agglutination.

Low Ionic Strength Solution (LISS)

• Increases antibody uptake during sensitization and the possibility of agglutination, in addition to lowering the zeta potential.

Polyethylene Glycol

• In LISS concentrates antibodies by removing water, to improve RBC sensitization, so centrifugation is not performed.

AHG Reagents

• Allow for agglutination of incomplete antibodies, polyspecific reagent contains antibodies to IgG and complement components.
• Monospecific antihuman globulin contains anti-IgG only, and is used to avoid investigations of insignificant antibodies.
• Any test that is negative after antihuman globulin reagent addition should be controlled, or else the screen must be repeated entirely

Gel Method

• The antibody screen can be done using a microtube filled with a dextran acrylamide gel. The reaction requires plasma and centrifugation. Omission of washing means fewer steps for the technologist.
• The process is stable for up to 24 hours, and reactions may be captured electronically, increasing productivity with standardized grading.

Solid Phase Adherence Method

• Utilizes solid phase adherence with RBC antigens coating microtiter wells. Incubation at 37°C allows present antibodies to react. Indicator red blood cells are added rather than any traditional AHG reagent.
• The solid phase adherence test is successfully automated and uses smaller sample volumes, ideal in pediatric settings with LISS, which changes color when sample is added.

Interpretation

• Agglutination or hemolysis at any stage signals the need for antibody identification studies.
• Whether reactions occurred and when they occurred in terms of phases must be considered. More than one sample reacting or not reacting also implies the condition.
• The nature of the reaction and the cell is also another determinant for the course of the antibody screening test.

Limitations

• Screening reagents are designed to detect clinically significant antibodies and avoid insignificant antibodies.
• A three-cell screen set gives 95% confidence that no clinically significant antibodies are present.
• A screening cannot detect low sensitivity or prevalence antigens and is affected by cell to serum ratios and other factors.

Cell-to-Serum Ratio

• The proper ration occurs at two drops of serum to one drop of red blood cell, may be altered when the antibody used.

Temperature and Phase of Reactivity

• Optimization is most easily done when performing pretransfusion compatibility, and performing tests before the immediate spin.

Length of Incubation

• Dependent upon the medium dependent testing must have a balance depending on time and antibodies.

pH

• Antibodies must be approximately neutral to work well.

Antibody Identification

• Testing is necessary to determine the clinical significance, using patients serum against red blood cells. Information concerning the patient may provide clues helpful in the antibody identification.
• A recent transfusion is a important clue to the antigen typing, and for a accurate assessment.

Reagents

• Panels of antigen should be diverse to distinguish antibodies. The antigen profile is lot specific and should not be changed
• The panel can show rare cells and positive and negative controls.

Exclusion

• Exclusion includes the ruling out of of antibodies that are not responsible, using RBC's for exclusion. One must only rule out with homozygous expression.

Evaluation of Panel Results

• This involves determining strength in phases, reacting in the same way, and examining each individually

Additional Techniques for Resolving Antibody Identification

Selected Cell Panels

• Simplest step by testing cells on a different panel with overlap in antigens. Also helpful when technologists determine known antibodies and presence.

Enzymes

• Help separate specificities and allow for identification if multiple antibodies are present. Ficin removes sialic acid reducing antibody activity.

Neutralization

• Other structures in the body have structures to bind with antibodies, like antigen structures.

Adsorption

• For auto-adsorption, autoantibodies are removed through adsorption techniques. If no antibodies are apparent, the procedure must be stopped. Also called Homologous Adsorption. If homologous adsorption is performed in place of autologous cells, the differential absorptions can employed. Also called Differential Adsorption.

Direct Antiglobulin Test and Elution Techniques

• Detection of antibodies in the blood tests, like hemolytic transfusion reactions, the direct test detects with vivo sensitization. Elution techniques are preformed after IgD and AHG reagents are used in tube methods and other testing methods. The methods are different depending on the intended outcome. Some commonly include temperature changing, and organic solvents changing the chemical properties

Antibody Titration

• Once an antigen is identified it is sometimes useful to quantify them. While, techniques like aminoassay are better, preforming an antibody titration is beneficial for determining antibody level. Twofold actions are done based on observed agglutination. After the titer is determined, follow the procedure accordingly based on observed samples.

Providing Compatible Blood Products

• The relative difficulty increases with clinical significance and decreased red blood cell survival.
• Other blood groups and examples are mentioned.

Case Studies

• Suspect multiple antibiodies when all or most cells are positive and show differing results. Use enzymes to solve. One tests one antibody and the other tests without.