1. MICROCYTOTOXICITY
( microlymphocytotoxicity)
Complement Dependent Cytotoxicity
(CDC)
By Dr Rania Abo-Shady
Ass. Professor of Clinical Pathology
Ain Shams University Fac. of Medicine
2. HLA - Typing
SEROLOGY used to be the ‘gold’ standard. Now being
superseded by molecular techniques as they become
more robust and time efficient.
CELLULAR rarely used now. Originally used for Class
II typing.
MOLECULAR fast becoming the method of choice.
Many laboratories test of choice.
3. SEROLOGY
HLA Typing is done serologically by MICROCYTOTOXICITY
which tests for complement mediated lysis of
peripheral blood lymphocytes with a standard set of
typing sera.
Micro-cytotoxicity assay, utilizes serum with known
anti-HLA antibodies that recognize particular HLA loci
(HLA-A, HLA-B, HLA-C, HLA-DQ, HLA-DR /not DP) in
order to match genetically similar individuals in hopes
of performing a tissue transplantation.
Viable peripheral blood lymphocytes are obtained by
density gradient centrifugation using Ficoll at 19º- 22ºC.
4. Cell isolation:
-For HLA Class I typing:
A mixture of T and B lymphocytes can be used by
lymphocyte separation .
-For HLA Class II typing:
B lymphocytes are required. (Normal population 85-
90% T and 10-15% B cells)
This can be achieved using a number of methods:
*Magnetic beads
Immunomagnetic bead separation is the current method of
choice.It utilises polystyrene microspheres with a magnetisable
core coated in monoclonal antibody for a HLA Class II b chain
monomorphic epitope. Positive selection.
*Nylon wool
In the past neuraminidase treated sheep red blood cell rosetting
and nylon wool have been used.
6. complement-dependent cytotoxicity
(CDC)
Lymphocytes are HLA-typed by crossmatching to panel
reactive antibodies (PRA) using the complement-
dependent cytotoxicity (CDC) test.
Complement
antibody
Positive reaction to antibody
kills cells. Dead cells pick up dye.
Buffy coat Negative reaction to antibody:
from patient cells survive and exclude dye.
7. Principle of Microlymphocytotoxic test
Microlymphocytotoxic test: 3 stages
• 1.Viable lymphocytes are incubated with HLA
specific antibodies. If the specific antigen is present
on the cell the antibody is bound.
• 2.Rabbit serum as a source of complement is added,
incubate. If antibody is bound to the HLA antigen on
the cell surface it activates the complement which
damages the cell membrane making it permeable to
vital stains.
• 3.Results are visualised by adding dye usually a
fluorochrome eg Ethidium Bromide although Eosin
Y have been used in the past.
8. Cells are prelabelled with Fluorochrome before
plating :
when cells are killed +ve test ,fluorescine leaks
out and a second fluorochrome a ethidium
bromide is added to visualize dead cells.( double
staining can be used by using a cocktail of
Ethidium Bromide and Acridine Orange)
If the reaction has taken place the EB or Eosin
enters the cell and binds to the DNA.
Test is left for 10 minutes and then read using an
inverted fluorescent microscope.
11. The percentage of stained cells
in each well is used to
determine whether the cells
are positive or negative for the Positive Negative
HLA antigen.
12. The Score Values in Lymphocytotoxicity Test (Terasaki and McClelland, 1964)
The percentage of
Score Evaluation
dead cells
0-10 1 Negative
11-20 2 doubtful positive/Negative
21-50 4 Weak Positive(++)
51-80 6 Positive(+++)
81-100 8 Strong positive(++++)
The number of dead (lysed) lymphocytes was compared
with the total number of cells quoted as score values
13. Advantages:
• Easily performed does not require expensive
equipment.
• Takes around three hours to perform
• Low level resolution, with good antisera
gives reliable results
Disadvantages:
• Requires large volumes of blood
• Requires viable lymphocytes
• Difficult to find good antisera for rarer
antigens in different populations
