Page |1
Laboratory 2: Neutralization Detected by Hemolysis
Skills: 15 points
Objectives
1. Using provided serum and diluent, carefully prepare a series of dilutions according to the
instructions provided.
2. Following instructions, evaluate tubes to determine the presence or absence of hemolysis.
3. After following all instructions, determine the endpoint of the procedure obtaining results falling
within ± 1 tube of instructor’s value.
4. Define “hemolysin” and “hemolysis”.
5. Appropriately record and report results as instructed.
6. State the principle of a neutralization procedure.
Introduction
Red blood cells are occasionally used in serological testing as indicators of antigen-antibody reactions.
The agglutination of red cells is the more common approach, but red cell hemolysis is also used. The
hemolysis of the red blood cell membrane can result as the consequence of the interaction of an antigen
on the red cell and the neutralization substance, the hemolysin. The term hemolysis is applied when the
red blood cell membrane is broken and hemoglobin is released from the cell. The supernatant of a
hemolyzed specimen will appear clear red, while the supernatant of non-hemolyzed specimens will be
colorless.
Principle
This test is to detect the presence of antibodies which have the ability to neutralize the hemolytic
properties of a hemolysin added to the test system. A specific amount of patient serum containing
antibody is added to a specific amount of hemolysin (antigen). If the antibody to the hemolysin is present
in the patient sample it will bind to the hemolysin. When red blood cells (RBCs) are added they will
NOT be hemolyzed since the hemolysin is bound up by the patient’s antibody. As the patient serum is
diluted out fewer and fewer antibody molecules will be available to bind to the hemolysin. Once enough
free hemolysin is present hemolysis of the red blood cells will occur.
The endpoint is the last tube showing NO hemolysis since this is the point at which sufficient patient
antibody was present to bind up all of the hemolysin. This is called a “neutralization” procedure because
the hemolysin will normally cause hemolysis of the RBCs. If the patient has an antibody to the hemolysin
and binds it up, no hemolysis can occur because the antibody has effectively “neutralized” the hemolyzing
affects of the hemolysin.
MLAB 1335 Hemolysin Titer Laboratory
Revised Fall 2014
Page |2
Laboratory 2: Neutralization Detected by Hemolysis
Materials
1. Patient serum
2. Hemolysin
3. 5% Red blood cell suspension
4. Five 12 x 75 test tubes and test tube rack
5. Three 1 ml serological pipettes and pipet bulb
6. 37°C heat block or water bath
7. Gloves
8. Timer
9. Sharpie marker
10. Serofuge
Procedure
1. Obtain patient sample from prep area.
2. Label five tubes with patient first and last initials at TOP of tube and number underneath.
3. Prepare the dilutions according to the following table.
Tube Number
1
2
3
4
5
Serum Amount
1.0 ml
0.8 ml
0.6 ml
0.4 ml
0.3 ml
Hemolysin
Amount
None
0.2 ml
0.4 ml
0.6 ml
0.7 ml
4. Shake to mix well and show your tubes to the instructor.
5. Add 0.5 ml of a 5% red blood cell suspension to each tube and note the appearance of the tubes.
6. Shake gently to mix and incubate at 37°C for 5 minutes.
7. Centrifuge all tubes for 2 minutes and examine supernatant for hemolysis.
Interpretation
The last tube with no hemolysis is the endpoint of the test.
MLAB 1335 Hemolysin Titer Laboratory
Revised Fall 2014
Page |3
Laboratory 2: Hemolysin Titer Recording/Interpreting Results
Name _____________________________________ Date _____________________
1. Record your observations in the following chart by putting “H” in the appropriate place(s) for
tubes showing hemolysis and “NH” for those without hemolysis.
2. According to the instructions, identify the tube that is the endpoint of the test and record it in the
appropriate place.
Patient Name
Identification Number
Tube 1
Tube 2
Test performed by
Tube 3
Tube 4
Date
Tube 5
Time
Endpoint is tube # _________________.
3. Explain in your own words the principle of this test procedure. Address the significance
of a “no hemolysis” result with “hemolysis” occurring.
Grade scale: Patient Name, Number (1 point each), tube results (2 points each), and initials/date/time (3
points each).
MLAB 1335 Hemolysin Titer Laboratory
Revised Fall 2014