3. Results

### 3.1 Calibration of the xytometer

Epics XL-MCL Cytometer (Coulter, Corp., Luston, UK) was checked and calibrated using the standard immunocheck particles (Becton Dickinson, Oxford, UK). The red cell samples automatically passed through the cytometer according to their FS and side scatter (SS). After defining the working conditions and the protocol to be used with the cytometer, it was determined that the most suitable conjugate dilutions were 1/50 and 1/100 (Figure 1). It was determined to work with 25,000 events at a flow rate of 600 events/s (Figure 2). The intensity of the signal observed in the negative controls is related to non-specific negative unions. There were no other phenomena related to false positives (Figure 3).

The most suitable conjugate was determined by processing 26 tubes in duplicate and 1/100 dilutions of the conjugate (Sigma Anti Human IgG -Fc Conjugate-F9512 specific and Kallestad FITC Conjugate #30446). Serial dilutions of commercial gamma globulin (250 μg/ml) were used as control. Figure 4 shows that the Sigma conjugate presents a greater fluorescence signal. After 10 assays using different fluorescence particles, the fluorescence intensity (MESF) and the fluorescence signal emitted by the cytometer presented good correlation. Figure 5 shows the linear relationship between the emitted MFI and the MESF. Peak's SD values in a range of 1.4–7.6 represent points with higher and lower intensity.

The histograms of the fluorescence parameters that were plotted according to the anti-Rho IgG concentrations can be seen in Figure 6. Figure 7a shows the working area determined with the forward and lateral dispersion of a homogenous population of non-sensitized cells. We observed no significant agglutination of auto aggregated red cells for anti-Rho IgG concentrations less than or equal to 960 ng/ml (4.8 IU/ml). We worked in intervals of ≤960 ng/ml to avoid increments of FS caused by auto aggregation (Figure 7b).

Figure 5.

Figure 6.

Figure 7.

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standard 68/419 960 ng/ml.

60 ng/ml AD, and 15 ng/ml AD.

Calibration curve of the flow cytometer, correlation between MFI and MESF.

DOI: http://dx.doi.org/10.5772/intechopen.89614

Flow Cytometry Assay for Quantitation of Therapeutical Anti-D IgG during Process Control…

FL1 histogram of fluorescence stockings obtained from seven dilutions of the WHO 68/419 standard using GR Rho + R1R1, 960 ng/ml AD, 480 ng/ml AD, 240 ng/ml AD, 120 ng/ml AD,

(a) Working region selected for Rho + R1R1 red blood cells according to the FS and SS of washed cells not sensitized in a concentration of 0.2% in PBS. (b) Increase of FS of GR R1R1 sensitized with anti-Rho IgG

Figure 2. Work and flow rate events.

### Figure 3.

Figure 4. Comparative curve of the conjugates.

Flow Cytometry Assay for Quantitation of Therapeutical Anti-D IgG during Process Control… DOI: http://dx.doi.org/10.5772/intechopen.89614

Figure 5. Calibration curve of the flow cytometer, correlation between MFI and MESF.

Figure 6.

aggregated red cells for anti-Rho IgG concentrations less than or equal to 960 ng/ml (4.8 IU/ml). We worked in intervals of ≤960 ng/ml to avoid increments of FS

Forward (FS) and side (SS) scatter graphs of a homogeneous population of non-sensitized cells.

caused by auto aggregation (Figure 7b).

Innovations in Cell Research and Therapy

Figure 3.

Figure 2.

Work and flow rate events.

Figure 4.

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Comparative curve of the conjugates.

FL1 histogram of fluorescence stockings obtained from seven dilutions of the WHO 68/419 standard using GR Rho + R1R1, 960 ng/ml AD, 480 ng/ml AD, 240 ng/ml AD, 120 ng/ml AD, 60 ng/ml AD, and 15 ng/ml AD.

### Figure 7.

(a) Working region selected for Rho + R1R1 red blood cells according to the FS and SS of washed cells not sensitized in a concentration of 0.2% in PBS. (b) Increase of FS of GR R1R1 sensitized with anti-Rho IgG standard 68/419 960 ng/ml.
