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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 cellmembrane is broken and hemoglobin is released from the cell. The supernatant of a hemolyzed specimenwill 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 patientantibody was present to bind up all of the hemolysin. This is called a “neutralization” procedure because the hemolysin will normally cause hemolysis of theRBCs. If the patient has an antibody to the hemolysin and binds it up, no hemolysis can occur because theantibody has effectively “neutralized” the hemolyzing affects of the hemolysin.

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 waterbath

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.

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 / Tube 3 / Tube 4 / Tube 5
Test performed by / Date / 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