1. Introduction

The Anti-D immunoglobulin (Anti-D IgG) produced from human plasma is used in the immunoprophylaxis of hemolytic disease of the newborn individuals who do not possess the red blood cells D antigen. Health authorities and manufacturers determine the concentration of antibody for each batch of medicine. Initially, the monitoring of Anti-D in the plasma of pregnant women and in finished products (Anti-D IgG) was performed by the anti-globulin test or Coombs test [1, 2] or by the continuous flow analysis of hemagglutination with a Technicon Autoanalyzer [3–8]. The automatic hemagglutination method has been considered as a reference by the European Pharmacopeia (EP); despite this, some groups of experts and regulatory bodies have replaced such methodology by radioimmunoassay (RIA)

[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 [18–20].

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

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 by the NIBSC expert group [16, 24].

2.4 Standard curve and statistical treatment

Calibration curve of the conjugate dilutions.

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

1.15 [25–28].

Figure 1.

3. Results

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3.1 Calibration of the xytometer

other phenomena related to false positives (Figure 3).

1.4–7.6 represent points with higher and lower intensity.

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

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

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

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

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
