The overall goal of the following experiment is to assess complement activation by antibodies against red blood cells or RBCs in patient serum. This is achieved by incubating red blood cells with patient serum in the presence of blocking anti C five antibody to induce C four and C3 deposition on the RBC surface. Next, the RBCs are stained with fluorescently labeled anti C four and anti C3 antibodies, and the amount of complement deposition on the RBCs can then be analyzed by flow cytometry in an alternative method.
The RBCs are incubated with patient serum in the absence of anti C five to induce complement mediated lysis. The percentage of lysed RBCs can then be determined by measuring the amount of hemoglobin released by the cells by spectrometry. The main advantage of this new technique over existing methods such as the hemolysis or the antiglobulin test used in routine diagnostics is that it is quantitative in nature and we can detect suboptimal differences in complement activation.
Elizabeth Brook postdoc in our lab will demonstrate procedures Begin by washing zero typed red blood cells three times with PBS, then incubate the pellet in 0.5%Roma lane solution at a one to two ratio for 10 minutes at 37 degrees Celsius after washing the cells three more times, store them as a 3%solution in fresh PBS at four degrees Celsius to analyze complement deposition on the RBCs by flow cytometry. First heat inactivate the desired amount of patient serum for 30 minutes at 56 degrees Celsius while the serum is being heat treated. Wash the brola treated red blood cells three times in Roal buffer after the final wash.
Resus suspend the pellet in vbg plus plus at a 0.5%dilution. Next at a glass pearl 25 microliters of 0.5%RBCs 37.5 microliters of fresh AB serum and 37.5 microliters of 200 micrograms per milliliter, anti C five to each well of a 96 well round bottom plate. Then add the heat inactivated patient serum to each well at the appropriate concentration supplementing with heat inactivated AB serum as necessary and including a negative control as well.
Use VBG plus plus to bring each well to a final volume of 150 microliters and then incubate the plate covered with E IA foil for one and a half to two hours at 37 degrees Celsius with shaking after the incubation. Wash the cells three times with PBS supplemented with 0.5%BSA, and then tag the cells with one microgram per milliliter. A fluorescently labeled anti C3 and anti C four monoclonal antibodies incubate the cells for 30 to 45 minutes at room temperature with gentle shaking, and then after washing the plate three times, resuspend the RBCs in 150 microliters of PBS plus 0.5%BSA per.
Well transfer the cell suspensions to a new 96 well round bottom plate, and then load the plate onto the flow cytometer, separate the single cells from the doublets using a forward scatter plot. Set the gate on the single RBCs and then select the appropriate detection channel for the fluorescent signals. Quantify the results using the median fluorescence intensity for quantitative hemolytic analysis, heat and activate the patient serum and wash the romaine treated RBCs as just demonstrated.
Then after incubating the RBCs with complement and patient serum as just demonstrated, but without the anti C five, spin down the cells for five minutes at 664 GS with reduced deceleration. Next, carefully transfer 90 microliters of supernatant to a new microtiter plate, taking care not to transfer the intact cells and to avoid creating air bubbles, and then measure the absorption at a four 14 to six 90 in a spectrophotometer express. The hemolysis as a percentage of the lysis of a sample of RBCs incubated in pure water.
In this first figure, representative scatter plots for brola treated RBCs are shown suitable gating for single RBCs can be seen here. Typically, around 95%of the RBCs fall within this single cell gate. In these histograms representative results for the effect of autoimmune hemolytic anemia or aha.
Patient serum on C four and C3 deposition are shown as expected, more patient serum leads to more complement deposition. Curiously, the complement deposition occurs in two distinct positive peaks. This is probably not caused by heterogeneity in the RBC population since the RBCs are drawn from a single donor, although the cause of this phenomenon is still being investigated, the median fluorescence intensities of the samples are plotted in these line graphs to show the reproducibility of the C four and C3 deposition assays.
Note the wide variation in the deposition of the various AH H patient samples. Finally, data from a hemolytic assay of an ahha patient serum sample containing auto antibodies to RBCs can be observed in these graphs. Titration with the serum sample yields a clear reproducible curve with a suitable complement inhibitor such as anti C five.
The hemolytic signal can be titrated away as demonstrated in this graph. After watching this video, you should have a good understanding of how to measure complement activation by red blood cell specific antibodies, either by flow cytometric analysis of C3 or C four deposition or by quantitative hemolytic assay.
