Objectives Define "agglutination"

MLAB 1335 Immunology/Serology

Laboratory 4: ABO Slide Agglutination Test
Objectives

1. 
   Define “agglutination”.
   
2. 
   Describe the two stages of agglutination.
   
3. 
   Define “hemagglutination”.
   
4. 
   Define “false positive” and “false negative” in ABO blood typing.
   
5. 
   List 4 limitations of the ABO slide agglutination procedure.
   
6. 
   Given a sample of red blood cells correctly perform the ABO typing procedure.
   
7. 
   Given the results of an ABO typing correctly interpret the results.
   

Introduction

Antigen-antibody reactions cannot be seen visually. Agglutination is a frequently employed technique in the Serology laboratory to detect antigens or antibodies. Agglutination is the clumping together in suspension of antigen-bearing cells, microorganisms, or particles in the presence of specific antibodies. In the test system a known antibody or antigen is added to an unknown antigen or antibody. If the substance being tested for (antigen or antibody) is present then agglutination will occur. Antigen or antibody may be artificially attached to latex particles or may be integral part of the indicator system being used to detect this reaction. In ABO typing, the A and B antigens are structures present on the surface of the red blood cells.

Agglutination occurs in two stages:

1. 
   Attachment of the specific antibody to the specific antigen - sensitization. This step is NOT visible.
   
2. 
   When antibodies then attach to a second antigen site on a different carrier molecule then lattice formation occurs which can then be seen visually.
   
   
   

This step results in a macroscopically visible reaction such as agglutination. When red cells are mixed with various reagent anti-seras (soluble antibody), agglutination will occur on the slides containing cells possessing the corresponding antigen. No agglutination will occur when the red cells do not contain the corresponding antigen. One primary application of this principle is blood typing. The process of using red blood cells (RBCs) as the indicator system results in hemagglutination. Hemagglutination is the agglutination of red blood cells. If the antigen to which the corresponding antibody is present on the red blood cells agglutination of the red cells will occur.
Principle

In this procedure a known antibody will be added to unknown antigens present on red cell. In the ABO slide typing known antibodies (anti-A and anti-B) are added to separate areas of a slide and mixed with the unknown red blood cells. If the antigen is present the antibody will bind causing agglutination which indicates the presence of the antigen on the red blood cell. If no agglutination occurs this indicates absence of the antigen to which the antibody is directed and no agglutination will occur. For example, if anti-A is added to a red cell that possesses the A antigen agglutination will occur indicating the presence of the A antigen. If no agglutination occurs when anti-A is added this indicates the absence of the A antigen.

Procedure'>Limitations of the Procedure

1. 
   Carefully dispense the red cell suspension and antibody in SEPARATE areas of the slide to prevent contamination of the reagent antibody or unknown red cell sample. Adding red blood cells directly to the drop of antisera may cause splash back, resulting in contamination of the red cells when the pipette is placed back in the RBCs suspension. If the red cells are used for other tests and the antigen to which the reagent antibody is directed is present this may result in agglutination of the red cell suspension causing a false positive. Conversely, if the anti-sera is dropped directly onto the red cells the red cells may splash back onto the dropper resulting in contamination and potential neutralization of the reagent antibody.
   
2. 
   The mixing of the antisera with the red blood cells should cover the entire bottom portion of the testing area on the slide. Inadequate spreading of the mixture will make interpretation difficult and may result in misinterpretation of results.
   
3. 
   If the slide is not tilted completely so that the reaction mixture goes to the end of the slide a false negative reaction may occur. A “false negative” is a reaction which gives a negative result when in fact the antigen being tested was present.
   
4. 
   It is critical to read the results at the end of the designated time. Allowing the reaction to go beyond the allotted time may result in the drying of the reactants, resulting in a false positive. A “false positive” is reaction in an antigen, is not present, yet agglutination occurs.
   

MLAB 1335 Immunology/Serology

Laboratory 4: ABO Slide Agglutination Test
Materials

1. 
   Anti-A typing sera
   
2. 
   Anti-B typing sera
   
3. 
   ABO Slides (1 per sample to be tested)
   
4. 
   Mixing stick (1 per sample to be tested)
   
5. 
   Disposable plastic pipettes (1 per sample to be tested)
   

Procedure

1. 
   On the section of slide labeled “anti-A” place one free falling drop of antibody A in the upper portion of the slide. NOTE: Hold the dropper one inch above the slide to ensure a full drop is dispensed. DO NOT touch the dropper to the slide.
   
2. 
   On the section of slide labeled “anti-B” place one drop of antibody B on the upper portion of the slide.
   
3. 
   Place one free falling drop of red cells to a separate area of each circle away from the drop of typing sera. DO NOT add it directly to the drop of anti-sera. Hold the pipette one inch above the slide to ensure that a full drop is dispensed.
   
4. 
   Carefully mix each solution with a separate side of the applicator stick, i.e., mix then flip applicator stick over and use opposite end. MAKE SURE THE ENTIRE SURFACE OF THE BOTTOM OF THE CIRCLE IS COVERED WITH THE MIXTURE.
   
5. 
   Tilt the slide slowly, back and forth, for two minutes. Make sure that with each tilt the antibody/RBC mixture falls complete to the end of the slide before tilting in the opposite direction. NOTE: You must use a timer.
   
6. 
   Record results as POS, if agglutination occurs or NEG, if there was no agglutination.
   
7. 
   The instructor will verify your results.
   

Interpretation

• 
  Agglutination (clumping) of the red blood cells is positive and indicates the presence of the antigen to which the antibody is directed.
  
  
  
• 
  No agglutination is negative and indicates absence of the antigen to which the antibody is directed.
  
  
  
• 
  It is critical to read the results immediately as false positives can occur when the mixture begins to dry on the slide.
  

Anti-A	Anti-B	Interpretation of Blood Group
POS	NEG	A
NEG	POS	B
NEG	NEG	O
POS	POS	AB

Name_________________________________ Date____________ Points /35
MLAB 1335 Immunology/Serology

Laboratory 4: ABO Slide Agglutination Test
Recording Results
Record your reactions for samples 1 and 2 in the spaces below. Write “POS” if agglutination was observed, write “NEG” if no agglutination occurred. Use the table on the previous page to interpret your results. Each section worth 2 points each, total is 20 points. Study questions 15 points.

Name	ID #	Anti-A	Anti-B	Interpretation	Instructor

1. 
   Define “agglutination”. ( 1 point)
   
   
   
2. 
   List and describe the two stages of agglutination and whether or not the reaction is visible.
   (4 points)
   
   1. 
      
      
   2. 
      
      
3. 
   Define “false positive” and list one cause. (1 point)
   
   
4. 
   Define “false negative” and list one cause. (1 point)
   
   
5. 
   List two limitations of the procedure. (2 points)
   
   1. 
      
      
   2. 
      
      
6. 
   If anti-B is added to a B cell describe what you would observe (use correct terminology) and the principle of WHY this would happen. (2 points)
   
   1. 
      Observation:
      
   2. 
      Why?
      

7. 
   If anti-A is added to an O cell describe what you would observe (use correct terminology) and the principle of WHY this would happen. (2 points)
   
   1. 
      Observation:
      
   2. 
      Why?
      

8. 
   Interpret the following reactions (2 points):
   

Anti-A	Anti-B	INTERPRETATION
POS	POS
NEG	POS

MLAB 1335

Revised Fall 2014