DEMONSTRATION OF COMMON IMMUNOHEMATOLOGIC REACTIONS

Precipitation

• Combination of a soluble antigen with a soluble antibody to produce an insoluble complex.

Agglutination

• The particulate antigen aggregates to form a large complexes, when a specific Antibody is present

Precipitation and Agglutination

• Both are visible expression of the aggregation of Antigen and Antibody through the formation of a framework

Agglutination

• Is the visible aggregation of particles caused by combination of specific antigen with its specific antibody

Agglutinogen

AGGLUTINATION

• _______________- Antigen that produces such reaction

Agglutinin

AGGLUTINATION

• _______________- Antibody that produces agglutination

Direct agglutination, Passive agglutination, Reverse passive agglutination, Agglutination inhibition, Co-agglutination

AGGLUTINATION

• Agglutination reactions can be classified into several distinct categories. What are those?

Sensitization phase and Lattice formation

2 step process of agglutination

Sensitization phase

• Antibody → Antigen with multiple determinants → Antigen-Antibody combination

• First reaction involves Antigen Antibody combination through single Antigenic determinants of the particle

• An individual Antibody will attach to a single antigen

• When the antibody and the specific antigen combines

• RAPID and IRREVERSIBLE

Lattice formation

• No visible reaction → Rearrangement → Visible agglutination

• Formation of cross link that form the visible aggregate

• Represent stabilization of the Antigen Antibody complexes

• One molecule can bind to a site on each of the two different cell

22 C or lower, 37 C

FACTORS AFFECTING AGGLUTINATION REACTION

SENSITIZATION PHASE

• TEMPERATURE- IgM (________), IgG (___________)

Zone of equivalence

FACTORS AFFECTING AGGLUTINATION REACTION

SENSITIZATION PHASE

• INCUBATION TIME- should be at ___________________________________

7

FACTORS AFFECTING AGGLUTINATION REACTION

SENSITIZATION PHASE

• pH- around ___

Low ionic strength

FACTORS AFFECTING AGGLUTINATION REACTION

SENSITIZATION PHASE

• Ionic strength- ___________________

  • RBC are negatively charge, so to disburse the ionic clouds that surround the RBC, we have to use a ____________________

Zeta potential

FACTORS AFFECTING AGGLUTINATION REACTION

LATTICE FORMATION

• _____________________- Force of repulsion between red cells. Because RBC are negatively charge, they tend to repel.

40:1

FACTORS AFFECTING AGGLUTINATION REACTION

LATTICE FORMATION

• Optimal concentration of antigen and antibody- The maximum of agglutination is best observed within the zone of equivalent.

• Serum to cell Ratio- ________

Effect pf centrifugation

FACTORS AFFECTING AGGLUTINATION REACTION

LATTICE FORMATION

• ____________________- The time and speed of centrifugation is important factor if you wanted to detect or to agglutinate the red cell.

• Purpose → Make the red cells closer together in the test environment

False positive result

Common causes of _____________________

• Contaminated equipment or reagents may cause particles to clump

• Autoagglutination

• Delay in reading slide reactions results in drying out of mixture

• Overcentrifugation causes cells or particles to clump too tightly

False negative result

Common cause of ______________________________

• Inadequate washing of red blood cells in antihuman globulin (AHG testing) may result in unbound immunoglobulins neutralizing the reagent

• Failure to add AHG reagent

• Contaminated or expired reagents

• Improper incubation

• Delay in reading slide reactions

• Undercentrifugation

• Prozone phenomenon (Need in the zone of equivalence)

Rouleaux formation

• is the arrangement of erythrocytes in groups that resemble stacks of coins. Rouleaux formation is associated with the presence of cryoglobulins.

• Mimics the agglutination reaction

• Red cells are causing an agglutination, due to ABNORMAL PROTEINS

• Result through the use of NSS

• Non-specific binding of red cells

positive

ROULEAUX FORMATION

• Can cause false ________ result

0.9%

ROULEAUX FORMATION

• REVERSIBLE, _____ NSS

Direct Agglutination

• Occurs when antigens are found naturally on a particle.

• Hemagglutination kits are now available for detection of antibodies to hepatitis A virus (HAV), hepatitis B virus (HBV), hepatitis C virus (HCV), and human immunodeficiency virus (HIV)

Widal Test Salmonella O

DIRECT AGGLUTINATION

• One of the best examples of direct agglutination testing involves known bacterial antigens used to test for the presence of unknown antibodies in the patient. (e.g. Widal Test- Salmonella O (somatic) and H (flagellar) antigens)

• ____________________- Rapid screening test to help determine the possibility of typhoid fever

Hemagglutination

DIRECT AGGLUTINATION

• If an agglutination reaction involves red blood cells

• The best example of this occurs in ABO blood group typing of human red blood cells, one of the world’s most frequently used immunoassays.

• If the particle that we are pertaining to is by the use of red cells

Direct Hemagglutination

DIRECT AGGLUTINATION

• Perform at room temperature without the use of any enhancement technique

Passive Agglutination

• Employs particles that are coated with antigens not normally found on their surfaces. A variety of particles, including erythrocytes, latex, gelatin, and silicates, are used for this purpose. The use of synthetic beads or particles provides the advantage of consistency, uniformity, and stability.

• have been used to detect rheumatoid factor; antinuclear antibody occurring in the disease lupus erythematosus; antibodies to group A streptococcus antigens; antibodies to Trichinella spiralis; antibodies to Treponema pallidum; and antibodies to viruses

• We need carrier particle

• It is used because the particle is not the antigen. It is serves as the carrier

Problem when we used Erythrocyte as the carrier particle:

PASSIVE AGGLUTINATION

• _________________________________________________________________- Include the possibility of cross reactivity, especially if there is Heterophile antibody.

Eryhtrocytes, latex particles, gelatin, silicates, bentonite, etc.

PASSIVE AGGLUTINATION

• The Carriers are??

7 micrometer, 0.8 micrometer

PASSIVE AGGLUTINATION

• Particles that we used vary: _______________, __________________

Tanic acid

PASSIVE AGGLUTINATION

• RBC making it possible to absorb particle: We are using ________. It makes RBC sticky for the absorption of an antigen.

Reverse passive agglutination

• antibody rather than antigen is attached to a carrier particle

• The antibody must still be reactive

• Rapid identification of antigens from such infectious agents as group B streptococcus, Staphylococcus aureus, Neisseria meningitidis, streptococcal groups A and B, Haemophilus influenzae, rotavirus, Cryptococcus neoformans, Vibrio cholera 01, and Leptospira.

• has also been used to measure levels of certain therapeutic drugs, hormones, and plasma proteins such as haptoglobin and C-reactive protein.

Rheumatoid Fever

• Passive agglutination

• It can cause false positive, because it reacts with any IgG antibody

AGGLUTINATION INHIBITION

• based on competition between particulate and soluble antigens for limited antibody- combining sites, and a lack of agglutination is an indicator of a positive reaction.

• Detect soluble Antigen

• Agglutination is a negative reaction

• Can detect either antigen and antibody

Cocaine, heroin

AGGLUTINATION INHIBITION

• Detection of HcG and illicit drugs such as _________ or ____________ are examples of very sensitive agglutination inhibition tests

Red blood cells

AGGLUTINATION INHIBITION

• Hemagglutination inhibition reactions use the same principle, except ______________ are the indicator particles. This type of testing has been used to detect antibodies to certain viruses, such as rubella, mumps, measles, influenza, parainfluenza, HBV, herpesvirus, respiratory syncytial virus, and adenovirus

Coagglutination

• is the name given to systems using bacteria as the inert particles to which antibody is attached.

• reagents have been used in identification of streptococci, Neisseria meningitidis, Neisseria gonorrhoeae, Vibrio cholera 0139, and Haemophilus influenzae.

Staphylococcus aureus, Protein A

COAGGLUTINATION

• ____________________is most frequently used, because it has a protein on its outer surface, called ________________, which naturally adsorbs the fragment crystallizable (FC) portion of antibody molecules.

Strong agglutination

SLIDE METHOD

• _____________-Large clumps and clear background

Weak agglutination

SLIDE METHOD

• _______________- small clumps and cloudy background

Negative

SLIDE METHOD

• ____________- even suspension and cloudy background

Hemolysis

• In an incompatible blood transfusion, antibodies in the recipient’s plasma bind to the antigens on the donated RBCs, which causes agglutination or clumping, of the RBCs.

• Agglutination is an antigen–antibody response in which RBCs become cross-linked to one another. When these antigen– antibody complexes form, they activate plasma proteins of the complement family. In essence, complement molecules make the plasma membrane of the donated RBCs leaky, causing __________ (rupture) of the RBCs and the release of hemoglobin into the blood plasma.

No hemolysis

INTERPRETATION

• __________________-Intact cell button with supernatant

Partial hemolysis

INTERPRETATION

• ___________________- Presence of cell button with pink supernatant

Complete hemolysis

INTERPRETATION

• _________________- Absence of cell button with red supernatant

Positive result

Partial hemolysis and Complete Hemolysis are ________________