MT 66 | BSMLS-3A-3D | A.Y.

2023-2024

OVERVIEW OF AGGLUTINATION
 Antigen-antibody reactions occurring in vitro (in laboratory testing) are detected by visible agglutination
of red cells or evidence of hemolysis.
 Negative reaction
 lack of antigen-antibody complex formation
 absence of hemagglutination
 Positive reaction
 formation of antigen-antibody immune complex
 presence of hemagglutination

TWO STAGES OF HEMAGGLUTINATION REACTION

A. First Stage: Sensitization Stage

 physical binding of antibody to an antigen on the red cell membrane
 no visible agglutination is observable at this stage
 Serum-to-cell ratio: ratio of antigen on the red cell to antibody in the serum

Overall effect of the serum-to-cell ratio: ↑ in Ab concentration → ↑ the binding probability

with the corresponding Ag

Factors Influencing First Stage of Agglutination

 Temperature of Reaction
 IgG antibodies → react at approximately 37°C

PREPARED BY: KAREN B. ROSETE, RMT 1

 MT 66 | BSMLS-3A-3D | A.Y. 2023-2024

 IgM antibodies → react at lower temperatures (room temperature or lower)

 Incubation Time
 Allows adequate time for the combination of antigen and antibody to attain equilibrium
 ↑ the incubation time → enhances detection of weak antibodies
 Immediate-spin step → test reagent + sample (with no incubation period)
 pH
 7.0 → optimal pH for hemagglutination (range: 6.5 – 7.5)
 Ionic Strength
 In an isotonic environment, the combination of antigen and antibody is hindered.
 ↓ ionic environment → ↓ shielding effect → ↑ amount of antibody uptake onto the red cell

B. Second Stage: Lattice Formation

 formation of lattice-type structure; composed of multiple antigen-antibody bridges between RBC
antigens and antibodies
 visible agglutination is observable

Factors Influencing Second Stage of Agglutination

 Distance Between Red Cells
 Zeta potential
• repulsive force between red cells in a physiologic saline solution
• red cells possess a net negative charge on the cell surface in a saline suspension; cations
(positively charged ions) from the saline environment are attracted to these negative
charges
• net negative charge surrounding RBCs is due to sialic acid molecules on the surface
of RBCs
• a stable cationic cloud surrounds each red blood cells and keeps the cells from adhering
to each other
 IgM antibody
• larger in size, has a pentamer shape, has multivalent properties
• efficient in causing RBC agglutination
• do not need enhancement media to strongly react with antigens
 IgG antibody
• Smaller in size, monomer, bivalent
• Visible agglutination might not occur

PREPARED BY: KAREN B. ROSETE, RMT 2

 MT 66 | BSMLS-3A-3D | A.Y. 2023-2024

• Needs enhancement techniques or potentiators to increase reactivity

 ↓ zeta potential → allows more positively charged antibodies to get closer to the negatively
charged RBCs → ↑ RBC agglutination (by IgG antibodies)

Potentiators or Enhancement Techniques for the Detection of IgG Antibodies

Reagent Action Procedure Type of Antibody ID
Saline 4–22°C (IgM) immediate Primarily IgM;
spin (IS) up to 60 min; IgG if incubated at 37°C
37°C (IgG) for 45–60 min
AHG Cross-links sensitized cells, 1. DAT: AHG added 1. Polyspecific; anti-
resulting in visible agglutination directly to washed IgG +
RBCs anticomplement
2. IAT: serum + 2. IgG monospecific:
screen cells; anti-IgG only
incubation at 37°C
for time
determined by
additive used; cell
washing before
addition of AHG
22 % Albumin* Causes agglutination by adjusting Incubation at 37°C for IgG
zeta potential between RBCs 15–60 min; cell washing
prior to IAT
LISS* Low ionic strength environment Incubation at 37°C for 5– IgG
causes RBCs to take up antibody 15 min; cell washing
more rapidly before IAT
PEG* Increases test sensitivity; Incubation at 37°C for 10– IgG
aggregates RBCs causing closer 30 min; cell washing
proximity of RBCs to one another before IAT
assisting in antibody cross-linking
Enzymes Reduces RBC surface charge; 1. One-step: Destroys Fya, Fyb, MNS;
destroys or depresses some RBC enzymes added enhances reactivity to
antigens; enhances other RBC directly to Rh, Kidd, P1, Lewis, and
antigens serum/RBC I antibodies
mixture
2. Two-step: RBC
pretreated with
enzymes before
addition of serum
*All additives should be added after the IS phase immediately before 37°C incubation
 Optimal Concentrations of Antigen and Antibody
 In ideal reactive conditions, an equivalent amount of antigen and antibody binds.
 Zone of equivalence
• Definition: number of binding sites of multivalent antigen and antibody are approximately
equal
 Prozone effect

PREPARED BY: KAREN B. ROSETE, RMT 3

 MT 66 | BSMLS-3A-3D | A.Y. 2023-2024

• Definition: when the concentration of antibody exceeds the concentration of antigen

• Solution: plasma or serum may be diluted with appropriate buffer
 Postzone effect
• Definition: when the concentration of antigen exceeds the concentration of antibody
• Solution: increase the number of antibodies to bind with each RBC by increasing the
serum-to-cell ratio
 Dosage effect
• Definition: It is the variation in antigen expression because of the number of alleles
present. Stronger agglutination is observed when a red cell antigen is expressed from
homozygous genes.
 Effect of Centrifugation
 Centrifugation decreases reaction time by increasing gravitational forces on the reactants

Summary of Factors Affecting Agglutination

Stage Factor Description
Sensitization Temperature IgG, 37° C; IgM, ≤22° C
Incubation time Immediate-spin or following
a specific time at 1-8° C, room
temperature, or 37° C
pH 7.0 (physiologic is ideal)
Ionic strength Can be adjusted with reagents
Lattice Formation Zeta potential Distance between cells caused by
charged ions
Zone of equivalence Antigen and antibody concentrations
Centrifugation Time and speed of
centrifugation to bring cells close
together
Temperature IgG, 37° C; IgM, ≤22° C
GRADING AGGLUTINATION REACTIONS
Principle: The presence of an antigen-antibody reaction is detected with red cell agglutination. The purpose of
grading reactions is to allow comparison of reaction strengths. This is beneficial in detecting multiple antibody
specificities or antibodies exhibiting dosage.

Materials:
1. Centrifuged serologic tests for agglutination
2. Agglutination viewer

PREPARED BY: KAREN B. ROSETE, RMT 4

 MT 66 | BSMLS-3A-3D | A.Y. 2023-2024

Procedure:
1. Gently shake or tilt the tube and re-suspend the red cell button in the tube. The tilt technique uses the
meniscus to gently dislodge the red cell button from the wall of the tube.
2. Observe the way that cells are dispersed from the red cell button.
3. Record reactivity by comparing the agglutinates to the descriptions below. The reactivity should be
assessed when the red cells have been completely re-suspended from the button.

Interpretation:

PREPARED BY: KAREN B. ROSETE, RMT 5

 MT 66 | BSMLS-3A-3D | A.Y. 2023-2024

Hemolysis as an Indicator of Antigen-antibody Reactions

 Activation of complement system – hemolysis along with agglutination occurs
 Red cell button – often smaller compared with the red cell button present in other tubes
 Supernatant – pinkish to reddish
 Hemolysis is detected in vitro using fresh serum samples.

MAIN REFERENCES:
❖ Harmening, D. M. (2012). Modern Blood Banking and Transfusion Practices (6 th Ed.). Philadelphia: F.A.
Davis Company
❖ Howard, P.R. and Blaney, K.D. (2013). Basic & Applied Concepts of Blood Banking and Transfusion
Practices (3rd Ed.). Missouri: Mosby

PREPARED BY: KAREN B. ROSETE, RMT 6