3.3 Standard curve and cutoff

To establish the standard curve, dilutions of WHO reference preparation were used in a concentration range of 30–480 ng/ml. The corresponding values of MFI were between 1.4 and 4.5. To establish the minimum level of detection, nonsensitized cells were run, with MFI values averaging 1.2 (Figure 9).

A linear and proportional to the log of the Anti-D concentration fluorescence response was obtained in the measured range. The linear regression of 270 points was r = 0.963. The linear measurement range was set between 120 and 240 ng/ml and was the most acceptable for our purpose.

Seven similar curves were run with each other (Figure 10a), and the average curve and two SD values were calculated at each point, and the upper and lower


limits of each reading were calculated and plotted. To determine the appropriate limits for our purpose, 19 standard curves were tested. As shown in Figure 10, at high concentrations of Anti-D, there are no significant deviations, and in the diluted samples, three showed deviations from the upper limit (0.24 μg/ml) and one below

(a) Determination of the lower and upper limits (------) and the values of 2DS for the mean standard curve. The limit lines were used for the approval of the standard curves. (b) From 19 standard curves, 15 were found

Standard curves using red blood cells of phenotype: R1R1 ( ), R1R2 ( ), R1r´ ( ), and R1r

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

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

Construction of the standard curve. A total of 18 curves was run and its linear regression was r = 0.963.

Figure 8.

( ).

Figure 9.

Figure 10.

59

completely within the lower and upper limit lines.

### Table 1. Red blood cell phenotype effect.

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

### Figure 8.

3.2 Effect of the red blood cell phenotype

Innovations in Cell Research and Therapy

and was the most acceptable for our purpose.

3.3 Standard curve and cutoff

Table 1.

58

Red blood cell phenotype effect.

Phenotypes R1R1, R1R2, R1r´, and R1r were tested to optimize the assay. The values of estimated concentrations (means-standard deviations-CV%) suggest that the tests are reproducible for each sample and each phenotype (Table 1). The R1R1 and R1R2 phenotypes presented a greater fluorescence signal, and, therefore, a greater amount of Anti-D was adsorbed than the rest of the phenotypes (Figure 8).

To establish the standard curve, dilutions of WHO reference preparation were used in a concentration range of 30–480 ng/ml. The corresponding values of MFI were between 1.4 and 4.5. To establish the minimum level of detection, non-

A linear and proportional to the log of the Anti-D concentration fluorescence response was obtained in the measured range. The linear regression of 270 points was r = 0.963. The linear measurement range was set between 120 and 240 ng/ml

Seven similar curves were run with each other (Figure 10a), and the average curve and two SD values were calculated at each point, and the upper and lower

ng STD 480 120 60 480 120 60 ng Rec. 480.45 119.70 60.30 478.75 118.00 60.10

Media 479.33 119.92 59.96 477.48 118.07 59.235 SD 2.39 0.84 0.36 0.86 0.95 1.56 VC% 0.5 0.7 0.6 0.2 0.8 2.6 SD + 2 484.12 121.59 60.69 479.21 119.97 62.35 SD 2 474.54 118.24 59.23 475.76 116.17 56.12

ng STD 480 120 60 480 120 60 ng Rec. 476.35 117.45 60.10 465.98 120.21 60.10

Media 473.95 117.675 58.46 473.47 117.64 57.96 SD 3.13 0.40 1.35 5.12 2254 1.51 VC% 0.7 0.3 2.3 1.08 1.9 2.6 SD + 2 480.21 118.49 61.17 483.72 122.15 60.98 SD 2 467.68 116.85 55.74 463.21 113.13 54.94

475.80 118.98 60.00 477.30 119.00 56.90 481.06 120.00 59.45 476.89 118.5 59.89 480.03 121.00 60.10 477.00 116.78 60.05

469.40 117.67 56.90 475.35 114.78 56.90 474.45 118.25 57.98 474.95 118.25 57.98 475.60 117.33 58.87 477.60 117.33 56.89

sensitized cells were run, with MFI values averaging 1.2 (Figure 9).

Phenotype R1R1 R1R2

Phenotype R1r´ R1r

Standard curves using red blood cells of phenotype: R1R1 ( ), R1R2 ( ), R1r´ ( ), and R1r ( ).

### Figure 9.

Construction of the standard curve. A total of 18 curves was run and its linear regression was r = 0.963.

### Figure 10.

(a) Determination of the lower and upper limits (------) and the values of 2DS for the mean standard curve. The limit lines were used for the approval of the standard curves. (b) From 19 standard curves, 15 were found completely within the lower and upper limit lines.

limits of each reading were calculated and plotted. To determine the appropriate limits for our purpose, 19 standard curves were tested. As shown in Figure 10, at high concentrations of Anti-D, there are no significant deviations, and in the diluted samples, three showed deviations from the upper limit (0.24 μg/ml) and one below the lower limit (120 ng/ml). The limits of 120–240 ng/ml were taken as validation criteria of the standard curve.

Inter-assay: (a) reference standard 68/419 WHO in ng/ml

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

\*Declared concentration for H: 150 μg/vial, P: 220 μg/vial, B: 164 μg/vial.

Accuracy and repeatability of standard (a) control samples (b).

Table 3.

Table 4.

cytometry.

61

Inter-assay: (b) Control samples

M1 M2 M3 M4 M5 M6 \_X

ng/ml MFI SD VC% +2SD 2SD

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

480 5.95 5.12 5.06 5.44 4.76 5.22 5.26 0.40 7.69 8.38 4.45 240 3.11 3.17 3.21 2.95 3.01 3.34 3.13 0.14 4.49 5.57 2.85 120 1.8 1.97 1.88 1.84 2.11 2.01 1.93 0.12 6.01 3.97 1.70 60 1.31 1.53 1.39 1.31 1.63 1.59 1.46 0.14 9.71 1.46 1.18 30 1.11 1.23 1.27 1.22 1.3 1.2 1.22 0.06 5.36 1.22 1.09

Product Gamma H\* Gamma P\* Gamma B\*

Dilution 1/250 1/500 1/750 1/350 1/700 1/1000 1/250 1/500 1/1000 MFI 1.21 1.16 1.05 1.37 1.24 1.06 1.21 1.15 1.02

Average MFI 1.19 1.161 1.05 1.35 1.22 1.06 1.215 1.14 1.01 SD 0.016 0,017 0,008 0.05 0.04 0.01 0.015 0.012 0.018 VC% 1.36 1.48 0.78 3.83 2.99 0.98 1.25 1.06 1.77 μg/ml 149.50 150.25 142.64 236.25 220.86 192.00 151.87 147.41 183.64 % Recovery 99.66 100.16 95.09 107.39 100.39 87.27 92.61 89,89 111.97 +2SD 1.23 1.20 1.06 1.45 1.29 1.08 1.24 1.17 1.04 2SD 1.16 1.13 1.03 1.25 1.15 1.04 1.18 1.12 0.97

Product n Average μg/ml Limit 95% VC% Declared concentration μg/ml % Recovery H 6 149.50 145–154 2.6 150 99.7

P 6 236.25 218–254 7.2 220 107.4

B 6 151.87 147–155 1.9 164 92.6

Comparison between anti-D concentration declared by the producer and concentration obtained by flow

6 150.25 146–155 3.3 100.2 6 142.64 140–144 1.3 95.1

6 220.86 208–233 5.7 100.4 6 192.00 189–196 2.1 87.3

6 147.1 145–151 2.7 89.9 6 183.64 176–189 2.7 111.9

1.18 1.16 1.04 1.39 1.24 1.06 1.23 1.15 1.03 1.19 1.17 1.04 1.39 1.25 1.06 1.19 1.13 0.98 1.22 1.19 1.04 1.38 1.23 1.07 1.22 1.14 1.00 1.20 1.14 1.06 1.3 1.15 1.05 1.21 1.16 1.01 1.18 1.15 1.05 1.27 1.22 1.04 1.23 1.13 1.02

The cutoff value set at 10 ng/ml (0.05 IU/ml) corresponds to a 1:1024 Anti-D standard (NIBS) dilution. After 135 plasma assays (55 Anti-D positive—80 negative), it was determined.

### 3.4 Reproducibility and repeatability

Tests in different matrices repeated 10 times simultaneously (intra-assay) during different days (inter-assay) evaluated the reproducibility and repeatability of flow cytometry. The sources of variation found were linked to the preparation of samples and variations in the cytometer (Table 2). The test showed good repeatability between tests, indicated by the coefficient of variation in representative samples (CV 4.95%, high concentration; 3.36%, intermediate concentration; and 4.78%, low concentration) (Table 3). These results coincide with Thorpe and Schäffner in the collaborative study of the European Union [13].

### 3.5 Recovery of Anti-D

The estimated concentration of Anti-D for each measured point in three commercial products presented a %CV in the range of 1–7.5% (Table 4). Two-thirds of the powers estimated by the manufacturers were within 95% of the acceptance limit of flow cytometry.

Only 1 of the 30 tests performed on 3 known concentration of commercial products showed deviations in the cut of the determined concentrations (87.3–111.9). The assay presented good recoveries of concentration in the samples tested (Table 4). The concentrations were estimated using the Wilcoxon test.


\*Commercial gamma globulins: concentration 1 = 204 μg/ml, 2 = 164 μg/ml; pool = 11 μg/ml, Cohn's Fraction II = 33 μg/ml.

### Table 2.

Accuracy and reproducibility of the standard (a) and control samples (b).

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


### Table 3.

the lower limit (120 ng/ml). The limits of 120–240 ng/ml were taken as validation

The cutoff value set at 10 ng/ml (0.05 IU/ml) corresponds to a 1:1024 Anti-D

Tests in different matrices repeated 10 times simultaneously (intra-assay) during different days (inter-assay) evaluated the reproducibility and repeatability of flow cytometry. The sources of variation found were linked to the preparation of samples and variations in the cytometer (Table 2). The test showed good repeatability between tests, indicated by the coefficient of variation in representative samples (CV 4.95%, high concentration; 3.36%, intermediate concentration; and 4.78%, low concentration) (Table 3). These results coincide with Thorpe and

The estimated concentration of Anti-D for each measured point in three commercial products presented a %CV in the range of 1–7.5% (Table 4). Two-thirds of the powers estimated by the manufacturers were within 95% of the acceptance limit

Only 1 of the 30 tests performed on 3 known concentration of commercial

(87.3–111.9). The assay presented good recoveries of concentration in the samples tested (Table 4). The concentrations were estimated using the Wilcoxon test.

Sample Pool of plasma\* Cohn Fraction II \* γ1\* γ2\* Concentration 12.5 30.2 197.2 179.5 n 10 10 10 10 Average MFI 2.08 11.26 3.12 3.49 SD 0.06 0.05 0.16 0.17 VC% 3.06 4.32 5.16 4.93 % Recovery 113.6 91.56 98.33 109.46 \*Commercial gamma globulins: concentration 1 = 204 μg/ml, 2 = 164 μg/ml; pool = 11 μg/ml, Cohn's Fraction II = 33

products showed deviations in the cut of the determined concentrations

Intra-assay (a) Reference standard 68/419 WHO in ng/ml Concentration 480 120 30 n 10 10 10 Average MFI 4.79 1.98 1.25 SD 0.24 0.07 0.06 VC % 4.95 3.36 4.78

Intra-assay (b) Sample control μg/ml

Accuracy and reproducibility of the standard (a) and control samples (b).

Schäffner in the collaborative study of the European Union [13].

standard (NIBS) dilution. After 135 plasma assays (55 Anti-D positive—80

criteria of the standard curve.

Innovations in Cell Research and Therapy

negative), it was determined.

3.5 Recovery of Anti-D

of flow cytometry.

μg/ml.

Table 2.

60

3.4 Reproducibility and repeatability

Accuracy and repeatability of standard (a) control samples (b).


### Table 4.

Comparison between anti-D concentration declared by the producer and concentration obtained by flow cytometry.
