Erythrophagocytosis Preparation

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-Aldrich 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.

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