MICROCYTOTOXICITY ( microlymphocytotoxicity)

Complement Dependent Cytotoxicity (CDC)

By Dr Rania Abo-Shady Ass. Professor of Clinical Pathology

Ain Shams University Fac. of Medicine

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 Now being superseded by molecular techniques as they become more robust and time efficient.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.

HLA - Typing

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.

SEROLOGY

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 beadsImmunomagnetic 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.

Coated with anti CD19 for B cells

complement-dependent cytotoxicity (CDC)

Lymphocytes are HLA-typed by crossmatching to panel reactive antibodies (PRA) using the complement- dependent cytotoxicity (CDC) test.

Complementantibody

Negative reaction to antibody:cells survive and exclude dye.

Buffy coatfrom patient

Positive reaction to antibodykills cells. Dead cells pick up dye.

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.

Principle of Microlymphocytotoxic test

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.

Terazaki plate and inverted microscopy

Negative (living cells) Positive (dead cells)

Negative Positive

The percentage of stained cells in each well is used to determine whether the cells are positive or negative for the HLA antigen.

Evaluation Score The percentage of dead cells

Negative 1 0-10

doubtful positive/Negative 2 11-20

Weak Positive)++( 4 21-50

Positive)+++( 6 51-80

Strong positive)++++( 8 81-100

The Score Values in Lymphocytotoxicity Test (Terasaki and McClelland, 1964)

The number of dead (lysed) lymphocytes was compared with the total number of cells quoted as score values

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

THANKS