2.3 Sensitization and immunoglobulin binding to the cell surface

0.2% red cell suspension was incubated with different test fractions (Anti-D standard IRP 68/419 OMS-NIBSC, human plasma, Fraction II of the Cohn process and gamma Anti-D globulin) in a 50/50 μl ratio. After stirring 30 min at 4°C, red blood cell suspension is washed with PBS-1% BSA and incubated with a 1/50 dilution of a goat anti-human IgG FITC (Figure 1). Red blood cells resuspended in 1 ml of PBS-1% BSA are measured with flow cytometry in Epics XL-MCL Cytometer-Coulter.

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

Figure 1.

[9–11] flow cytometry and a competitive enzyme immunoassay (EIA). These alternatives to the current reference method of European Pharmacopeia have been successfully investigated during international collaborative studies [11–17]. Competitive flow cytometry and EIA assays have been described previously

Cohn-Oncley process, and finished product) [13, 14, 21–23].

by the NIBSC expert group [16, 24].

Innovations in Cell Research and Therapy

2.1 Description of the flow cytometry test

2. Materials and methods

concentration.

PBS—pH 7.2.

Cytometer-Coulter.

54

We describe the use of flow cytometry in the control of manufacturing processes of Anti-D globulin, establishing the validation parameters and compliance with the quality standards required by the GMP and established by the producer at the different stages of the industrial process (hyperimmune plasma, Fraction II of the

The rationale of the technique lies in the binding antigen antibodies, which are labeled with fluorescein detected by flow cytometry. The Anti-D immunoglobulin is quantified in comparison with the international reference preparation, calibrated in international units (IU), which allows to give a specification in IU/ml [20, 24]. In relation to flow cytometry, Expert Group No 6B of the NIBSC standardized a procedure to be applied in the evaluation of Anti-D in Anti-D IgG solutions [24]. In our laboratory, a flow cytometry technique was developed for the quantititation of Anti-D antibodies of the Gamma-Rho UNC, designed according to the procedures described in the literature [20, 24] and validated according to the criteria indicated

A laser hits the red fluorescein-labeled cells that cross the cytometer in a continuous flow. The light emission produced by the interaction is detected by the forward scatter (FS) and converted into a voltage pulse (FS). A limit of 25,000 events is determined at a flow rate of 600 cells per second with a power of 1300 V. The concentration of Anti-D will be proportional to the size and intensity of fluorescence emitted by the antigen-antibody fluorescein complex of each red blood cell. The mean fluorescence intensity is plotted according to the logarithm of the Anti-D

2.2 Preparation of cell suspension and Anti-D IgG samples and sensitization

A Rho red cell suspension (Red Cell phenotypes R1R1/R1R2 and Rh-rr as control), were diluted to 0.2% in phosphate-buffered saline (PBS) pH 72 and plasma samples, Fraction II and finished product were diluted 1/5 to 1/100 with 1%

0.2% red cell suspension was incubated with different test fractions (Anti-D standard IRP 68/419 OMS-NIBSC, human plasma, Fraction II of the Cohn process and gamma Anti-D globulin) in a 50/50 μl ratio. After stirring 30 min at 4°C, red blood cell suspension is washed with PBS-1% BSA and incubated with a 1/50 dilution of a goat anti-human IgG FITC (Figure 1). Red blood cells resuspended in 1 ml

2.3 Sensitization and immunoglobulin binding to the cell surface

of PBS-1% BSA are measured with flow cytometry in Epics XL-MCL

and immunoglobulin binding to the cell surface

[18–20].

Calibration curve of the conjugate dilutions.

### 2.4 Standard curve and statistical treatment

In a concentration range of 1500 ng/ml (7.5 IU/ml)–180 (0.9 IU/ml) ng/ml of international reference for human Anti-D immunoglobulin preparation (IRP 68/419 WHO), we establish the standard curve. No Anti-D sensitized cells were used, to set false positives and the minimum level of detection. Mean fluorescence intensity values were between 600 and 1900 and averaged around 800. The upper and lower limit values were determined using five standard curves. Nonparametric statistics was used and processed with Microsoft Excel 7.0 program and Method Validator 1.15 [25–28].
