Definitions & Factors of Agglutination

Question 1

Agglutination

Answer 1

Clumping together of RBCs or any particulate matter resulting from interaction of antibody and corresponding antigen

Question 2

Agglutinin

Answer 2

Antibody that agglutinates cells or other particulate matter

Question 3

Agglutinogen

Answer 3

A substance that stimulates the production of an agglutinin
Acts as an antigen

Question 4

Two Stages of Sensitization

Answer 4

Stage 1: Sensitization
- Antibody Attachment
- Equilibrium constant
Stage 2: Lattice formation
- Formation of bridges between sensitized cells

Question 5

Factors Affecting Sensitization

Answer 5

Specificity is Primary Determinant (Type of Antibody)
- IgM (better lattice formation) or IgG
pH
Temperature
Incubation Time
Ionic Repulsion
Zeta Potential

Question 6

Antigen-Antibody Ratio

Answer 6

Affects speed of antibody uptake
Increased Ab amount = Increased sensitivity

Question 7

pH

Answer 7

Optimal pH
Routine Testing 7.0 (6.5 - 7.5)

Question 8

Temperature

Answer 8

IgM perform best below 37 C (not major concern)
IgG perform best when at 37 C
Ab Identification designed to cover a range (22-37 C)
- ensure identification of any possibly present ab
- Ab reacting below 37 C are considered clinically insignificant

Question 9

Incubation Time

Answer 9

Time allows for antibody uptake
Example - Rh antibodies
- 25% taken up in first 15 minutes
- 75% taken up in the first hour

Question 10

Ionic Repulsion

Answer 10

Created by Na+ and Cl- ions
Low Ionic Strength Saline (LISS) used to minimize effect
- gets around negative charge on RBC surface

Question 11

Zeta Potential

Answer 11

Occurs due to Net Negative Charge on RBCs
- From Sialic Acid
“Ionic Cloud”

Question 12

Alteration of Zeta Potential

Answer 12

Suspending Medium can mitigate effect
- Albumin (problem as causes spontaneous agglutination)
- LISS (helps to identify specific ab groups better)
- Polybrene & Polyethylene Glycol (PEG)

Question 13

Factors Affecting Lattice Formation

Answer 13

Enzymes
Antihuman Globulin
Additional Reagents

Question 14

Enzymes

Answer 14

Decrease surface charge by cleaving surface proteins
Remove obstructions
Frequently used enzymes:
- Papain (from papaya)
- Ficin (from figs)
- Bromelin (from pineapple)
Effect - reduction of surface charge
Removes antigen binding sites: M, N, S, s, Fya, Fyb

Question 15

Antihuman Globulin

Answer 15

Antibody to human antibody/complement
Detects cells coated with antibody, complement, or both
Crosslinks “coated cells”

Question 16

Additional Reagents

Answer 16

Dithiothreitol (DTT)
2, mercaptoethanol (2 ME): destroys IgM and leaves IgG
ZZAP: dissociates IgG from RBC, alters RBC surface charge