HEMOLYSIS, CFT, AND NEUTRALIZATION • We will see hemolysis if there is no antibody

present in the patient sample, thus the

HEMOLYSIS complement is free and it will bind with the
• Ruptured or lysis of Red Blood Cells. coated sheep red blood cells, and thus
o Due to antigen and antibody cause hemolysis.
reaction, and complement fixation.
• Hemolysis is one of the most observable COMPLEMENT FIXATION TESTING RESULTS
laboratory results in Immunology and NEGATIVE= Presence of Hemolysis; no antibodies in
Serology laboratory. the patient’s sample.
COMPLEMENT FIXATION TESTING POSITIVE= No Hemolysis; antibodies are present in
• Uses reagent with complement the patient’s sample.
• Indicator of the presence of either antigen NEUTRALIZATION
or antibody • Can be used only when the antibody being
• 2-stage process: measured is directed against a hemolysin
o A test system with antigen and • Measures the ability of the patient’s
antibody, one of which is unknown antibody to neutralize infectivity and protect
o An indicator system consisting of cells from infection
sheep red blood cells coated with • Gold standard for assessment of protective
hemolysin, which will lyse in the antibody
presence of the complement. • It is not used for routine diagnosis, but is
Hemolysin is a bacterial toxin confined to specialty or public health
capable of directly lysing a red cell. laboratories for special indications.
So, this is a substance such as
Streptolysin O, Streptolysin S, which
is produced by most group A strains
of streptococci that disrupts the
membrane integrity of red blood
cells causing the release of
hemoglobin.
• May be used to detect a specific antigen or
antibody. Complement itself can actually be
used as a reagent in the test known as the
Complement Fixation
• This technique has been used in the • Here we can see that there is no antibody
detection of viral, fungal, and rickettsial present, and we added a hemolysin or toxin
antibodies. molecules. What will happen here is that the
hemolysin will attack the red cells or it will
disrupt the membrane of the red cells, and
then we will see hemolysis.
• If there’s the presence of antibodies in the
patient’s serum, and we add toxin
molecules/hemolysin. In this case, the
antibodies will bind to the hemolysin. Thus,
the hemolysin is no longer available to
disrupt the membrane of the red cells.
Hence, we will not see hemolysis if there’s
the presence of antibodies.

• This is what happens in complement fixation

testing. So, what we do is we mix the
patient’s serum, which contains the
antibodies and a reagent which contains the
antigen (see image above for further
explanation).
 PROCEDURE 6. After this, we check our tubes for hemolysis:
1 Prepare 6 test tubes to be used for fresh serum a. If it is colorless and clear
(label as 1A to 6A) and 6 test tubes to be used supernatant we report is as low
for aged serum (Label as 1B to 6B) hemolysis
2 Add corresponding volume of sample: b. If we see pink or faint red
supernatant, we report is partial
hemolysis
c. If we see that the supernatant is red,
that means that we should report it
as complete hemolysis.
7. For tubes 1A and 1B, what will be the reporting
or expected results? For 1A and 1B the
expected results are no hemolysis.
Why is there NO hemolysis?
3 Add 100 uL of 3-5% red cell suspension to each • Because tubes 1A and 1B does not contain the
tube serum. It only contains the red cell
4 Mix and centrifuge for 30 seconds at 2,500 suspension. So, we do not expect any
RPM reaction to occur.
5 Carefully take out the tubes from the centrifuge • For the other tubes, we have to observe the
and observe the supernatant. Record and supernatants, some tubes have faint red
interpret results. supernatants which is reported as partial
MANNER OF REPORTING hemolysis, and there are no tubes that are
1 No hemolysis- colorless and clear supernatant completely red and with no cell button, so we
2 Partial hemolysis- pink or faint red supernatant report it as complete hemolysis if we no
3 Complete hemolysis- red supernatant longer see red cells, that means that the
LAB DEMO contents of the tube is clear and red, which
Preparation of 6 test tubes for fresh serum means complete hemolysis.
o Faint red supernatant – partial
1. Prepare 6 test tubes to be used for fresh
hemolysis
serum (label as 1A to 6A)
o No red cells + contents or tube is clear
2. Add the corresponding volume – add fresh
and red – complete hemolysis
serum from 1A to 6A using micropipette
Tube
• Most tubes only show partial hemolysis.
A1 A2 A3 A4 A5 A6
No. Others, no hemolysis at all since their
Fresh
0 50 100 150 200 250 supernatant is clear.
serum
*Adjust the micropipette to get the correct amount of uL of fresh
serum. The tip must be replaced in each serum
*add fresh serum from 1B to 6B using micropipette.
FRESH SERUM VS. AGED SERUM
Tube
1B 2B 3B 4B 5B 6B
No. FRESH SERUM
Fresh
0 50 100 150 200 250 •Comes from freshly collected blood and contains
serum
viable antibodies and viable complement.
3. Add 3-5% red cell suspension to each of the
•Since antibodies and complements are still present,
test tubes. Make sure that your red cell
there will be reaction like agglutination, and
suspension is well mixed. In here we make
hemolysis.
use of 5% red cell suspension. We add 100uL
AGED SERUM
of the 5% red cell suspension to each of the
• Old, unpreserved, or long standing in room
test tube.
temperatures (for more than 24 hours). The
4. After the addition of the red cell suspension
antibodies are no longer viable, and complement is
to all of the test tubes, mix the contents of
no longer working. Thus, there will be no reaction
each tube. You can do this by gently tapping
once we add the corresponding antigen.
the test tubes. We do mixing before we
centrifuge our test tubes.
5. Centrifuge for 30 seconds at 2500 rpm. After
centrifugation, gently get your tubes from the
centrifuge. Make sure that you do not disturb
so you’ll be able to observe the supernatant.
Since we are observing hemolysis, we have to
make sure that we see the supernatant
properly. Get your tubes gently.