Antigen-Antibody Reactions

4+ reaction in tube

Single clump of cells, clear background.

3+ reaction in tube

Large clump of cells plus some small clumps, clear background.

2+ reaction in tube

Medium small clumps of cells, background may be cloudy.

1+ reaction in tube

Small clumps of cells, cloudy background.

Neg reaction in tube

No agglutination; free flowing cells.

4+ reaction in gel

Single layer of cells a the top of the gel column, no cells trailing down into the column.

3+ reaction in gel

Majority of cells trapped at the top of the gel column with some cells or agglutinates trailing down through the top half of the column.

2+ reaction in gel

Cells and agglutinates distributed through the column.

1+ reaction in gel

Small agglutinates mostly in the bottom half of the gel column, may be a cell pellet at the bottom.

Neg reaction in gel

Pellet of cells at the bottom of the gel column.

Dosage effect

A significant difference in antibody reaction strength depending on the quantity of the target antigen present on a target red blood cell.

Monoclonal antisera

Made using identical immune cells that are all clones of a specific parent cell. They recognize only a single epitome of an antigen and is extremely specific. Each lot is identical.

Polyclonal antisera

Made using several different immune cells. Will have the affinity for the same antigen but different epitopes. May have lot to lot variance.

Polyspecific AHG

Contains BOTH Anti-IgG and Anti-C3d. Anti-IgG binds to exposed Fc fragment of bound IgG molecules, while Anti-C3d detects complement fragment due to IgM binding or other form of complement activation happening. This reagent is preferred for the DAT.

Monospecific AHG

Contains EITHER Anti-IgG or Anti-C3d. Monospecific IgG is required for the IAT involved in antibody screen procedures.

AHG

Checks for antigen-antibody sensitization that did not lead to lattice formation. It forms a “bridge” when antigen-antibody complexes are present. Used for the IAT and DAT.

Indirect antiglobulin test (IAT)

Demonstrates in vitro reactions between red blood cells and IgG antibodies. Detects unexpected antibodies.

Direct antiglobulin test (DAT)

Detects red blood cells that have been sensitized in vivo (antibodies attached to RBCs).

Causes of false negative results in both IAT and DAT

• Inadequate washing

• Interruption in testing

• Deteriorated or neutralized AHG or AHG not added

• Improper concentration of RBC

• Improper centrifugation

Causes of false negative results only in DAT

• Deterioration of complement

Causes of false negative results only in DAT

• Deterioration of complement

Causes of false positive results in both IAT and DAT

• Dirty glassware

• Contaminated saline or AHG

• Cells agglutinating prior to AHG addition

• Improper reading technique

• Improper centrifugation

Causes of false positive results only in IAT

• Preservative-dependent antibody in LISS reagent

• Cells with positive DAT

Causes of false positive results only in DAT

• Clotted or refrigerated specimen

Antibody screen

Detects antibodies to red blood cell antigens

Antibody identification

Identifies the specificity of red blood cells antibodies.

Crossmatch

Determines serologic compatibility between donor and patient prior to transfusion.

Antigen typing

Identifies a specific antigen on a patient or donor’s red blood cells.

Gel technique

Where antigen-antibody reactions occur at top of column, agglutinates will become trapped in gel during centrifugation. Designed to detect IgG.

Solid phase methodology

Red blood cell antigens/antibodies coated to the bottom of microliter plate wells. Agglutinates will cause diffuse RBC pattern.