Fast and Specific Assessment of the Halogenating Peroxidase Activity in Leukocyte-enriched Blood Samples

The overall goal of this erythrocyte depletion method is to achieve a quick and simple leukoctye-enrichment from a small blood sample. Many advantages of this technique are that it is cheap, reliable, and fast. Moreover, it can be applied to human and non-human blood samples.

Before beginning the lysis procedure, adjust the pipette for water administration to two milliliters, and the pipette for PBS to five milliliters. Then place open flasks of water and PBS under a laminar flow hood at room temperature. As the hypotonic lysis is a time-critical process, everything should be prepared previously.

Moreover, the lysis proceudre should always be done in the same way, to have compatible results. Next, transfer 100 microliters from each blood sample into a 15 milliliter conical tube. And add two milliliters of distilled water to each sample with vortexing at the medium setting.

Rest the cells for 60 seconds, then add five milliliters of PBS. And mix the samples by vortexing. Now, collect the cells by centrifugation, and decant the supernatants.

Invert the tubes and tap the openings on a paper towel to remove as much liquid as possible. Then repeat the hypotonic lysis procedure as just demonstrated. After obtaining a dry pellet for the second time, re-suspend each sample in 500 microliters of HBSS, until a homogeneous solution is obtained.

And transfer the cells into 1.5 milliliter, micro-centrifuge tubes on ice. To check the peroxidase specificity of the samples after amino phenol fluorescein, or APF staining, first add five microliters of the heme peroxidase inhibitor, 4-ABAH, to the appropriate samples for a 15 minute incubation at 37 degrees Celsius. Next, add five microliters of APF working solution to each tube, and gently vortex the cells on the medium setting.

Incubate all of the samples at 37 degrees Celsius for 30 minutes. Then add five microliters of hydrogen peroxide working solution to each sample, and gently vortex the tubes. After 60 minutes at 37 degrees Celsius, centrifuge the cells, and carefully remove the supernatant without disturbing the pellets.

Finally, re-suspend the cells in 250 microliters of HBSS, and store the samples in the dark until their analysis, to avoid photo bleaching. Here, a representative example of the leukocyte populations after hypotonic lysis and APF staining as just demonstrated is shown. By plotting cell size versus granularity, the robust depletion of the erythrocytes can be observed, with the red blood cell and debris population accounting for only 22%of the events.

Further, the intensity distribution of the APF-derived fluorescence facilitates a clear discrimination of the peroxidase-negative erythrocites and lymphocytes, with a moderate APF oxidation observed, for the peroxidase-positive monocytes and eosinophils, and a robust peroxidase oxidation exhibited by the neutrophils. Once mastered, this technique can be applied to up to 50 blood samples in parallel if it is performed properly. Following this procedure, other methods like antibody staining can be performed to identify the cell subsets of interest.

After its development, this technique paved the way for researchers in the field of small animal studies to explore the role of halogenating activity of MPO and EPO in animal models for chronic inflammatory diseases.