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Erythrophagocytosis Assay

Document ID: DQ068
Relevant to following products: WorkOut 2.0 Manta
Document description: Application Example: Chemiluminescence Assay for Erythrophagocytosis

A luminol-dependent chemiluminescence assay for the assessment of the phagocytosis of erythrocytes sensitised with IgG immunoglobulin by mononuclear leukocytes.

Reaction mixture consists of mononuclear leukocytes, luminol and erythrocytes (either IgG sensitized or unsensitised controls).

Luminol (5-amino-2,3-dihydro-1,4-phthal-azinedione), Sigma Chemical Company.

Positive and negative controls are included in each assay in duplicate.

The Positive Control: Rh D Positive cells (R1r) sensitised with anti-D.
The Negative Control: Rh D Positive cells (R1r) incubated with AB serum.
Test samples: Test red cells sensitised with patient sera.
All test samples are assayed in duplicate with their own single blank control (red cells sensitised with AB serum not patient sample).

Temperature required: 37C

Each assay is run for 150 minutes with light emission for each cuvette (25 in total) being measured approximately every 4 minutes for 37 cycles. Continuous mixing should be switched on for the first cycle only then switched off.

Peak luminescence is seen between 35-45 minutes, the reaction being exhausted by 150 minutes.

When the assay is completed, calculate the area under each output curve. This is a measure of the CL response generated. The mean area underneath the output curves is calculated for each test sample and used in the calculation of a ratio.

Opsonic Index (OI) = Mean area under curve of sample / Mean area under curve of its blank control

An opsonic index < 1.2 is indicative of a negative result.

The test sample identified as the positive control must have an ipsonic index greater than 2.0 (OI= mean area under Positive Control/ mean area under Negative Control) in order to verify that the assay has been successful and the results are acceptable.

Downing I., Templeton J.G., Mitchell R. & Fraser R.H. (1990). A Chemiluminescence Assay for Erythrophagocytosis. J. Bioluminescence and Chemiluminescence, 5, 243-250.

To set-up:

Set-up readings with the Generic Wizard.
Create a microplate layout as follows:

Add an Integral transform
Add a Matrix Expression transform and enter the expression:


Set the Output Matrix name to Opsonic Index. This expression finds the average of each well with the well beneath it.

Create a single Cut-Off transform and enter 1.2 as the cut-off point. (The input matrix is Opsonic Index.)

Create a Validation Expression transform and enter:

Pos Control1 > 2.0

The input matrix is Opsonic Index.

Example results file available for download here.

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