ANTIGEN ,

ANTIBODY CHARACTERISTICS

& REACTIONS

Characteristics of an Antigen
> Must be foreign to the body

> Must possess a high molecular weight

> Must gain entry to the animal body

> Must possess high order of specificity

> Must possess chemical complexity

Characteristics of an Antibody
> Specificity

> Heterogeneity
> Recognition to oneself > Memory

Factors Affecting Ag-Ab Reactions

1. Complete and Incomplete Antibodies

Immunoglobulins Involved in Immunohematologic reactions

IgG disulfide bond; 14nm IgM pentameric / pentamer, first line of defense because they are the first one to be produced because of its antigenic structure; 35 nm **IgA monomeric lines with mucosal surface and predominantly is in with secretion

Characteristics of IgG and IgM

Varying Lattice fromation

2. Effect of Surface charge

3. Pretreatment of Red Cells

> to reduce the Zeta Potential
-

Protamine ( hexadimethrine bromide) Polystyrene

4. Use of Protein Media

> To reduce the zeta potential

Enzyme : bromelin . 0.5% , pH 5.5 ficin & trypsin 0.1% , pH 7.3 Papain ..0.1% , phH 6.5

5. Use of Antihuman Globulin

6. Chemical Reduction of IgG 7. Effect of pH 8. Effect of Temperature

4 degrees to < 37 degrees (cold reacting Ab) 37 degrees centigrade (warm reacting Ab)

Note: Abs detected at 37 degrees Centigrade are significant

9. Effect of Ionic Strength

Short incubation period needed LISS (0.2% NacL and 0.7% Glucose)

10. Effect of Cell Dosage

Heterozygous production AO ---- A antigen AB ---- A , and B antigens BO ---- B antigen Homozygous production AA ----- A antigen BB ----- B antigen

11. Effect on the Location of the Antigen

Considerations when reading Agglutination Reactions

1. Hemolysis

> All patients must be O

> False negative Occurs before addition of reagents (Ag or Ab) How to check? Centrifuge the mixture, if none settled : Positive

Causes of Hemolysis
> Excessive pulling of plunger during blood collection > Using hypotonic solution during red cell preparation

Red Cell Suspension 2% 5 %

2. Rouleaux Formation

> Stack of coin

Falsely Interpreted Reactions

1. Sample Problems
> Plasma / Serum Serum contain fibrin strands : not completely coagulated Elevated amounts of Proteins and Presence of abnormal Proteins > Red Cells coated in-vivo : IAHA, TR, HDN

Importance of Mixing Insufficient or delayed mixing of serum tubes may result in delayed clotting. Inadequate mixing of anticoagulant tubes may result in platelet clumping, clotting or incorrect test results.
http://us.gbo.com/preanalytics/admin/modules/document_repository/docs/b0a06bd67

2. Reagents Used
> Degraded potency
Improper storage

Proper storage : 2 8 degrees Centigrade

> Concentration of Saline Solution

Hypotonic or Hypertonic Proper concentration : 0.85% to 0.90%

> Antisera contain other antibodies

3. Equipment Problems
> Incorrect calibration of centrifuge machine > Contaminated glassware used

4. Technical Errors
> Commonly done by MedTechs

CAUSES OF FALSE POSITIVE REACTIONS IN AGGLUTINATION Causes

Over centrifugation Contaminated glassware Autoagglutination Cross reactivity

Remedy
Regulate speed and time Keep supplies covered and stored in a clean place Use control and Saline Use purified reagents and monoclonal Ab

Presence of Heterophil Abs

Delayed reading slide test Too heavy Red cell suspension

Use pre absorbed serum and

Read results immediately Prepare Red Cell Suspension properly

CAUSES OF FALSE NEGATIVE REACTIONS IN AGGLUTINATION Causes

Under centrifugation Inadequate red cell washing Reagents not active Delays in carrying out the procedure Insufficient incubation time and temperature Too light red cell suapension Failure to add AHG Prozone phenomena

Remedy
Regulate speed and time Wash red cells properly Proper storage of reagents Once a procedure is begun, follow through until the end without delay Observe proper time and temperature Prepare red cell suspension properly Monitor the addition of reagents Dilute the serum properly and repeat the test

Teach us to number our days aright that we may gain wisdom.

Psalm 90:12