Journal of Immunological Methods 422 (2015) 95–101
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Research paper
Validation of Single Radial Haemolysis assay: A reliable method to measure antibodies against influenza viruses Claudia Maria Trombetta a,1, Daniele Perini b,1, Licia Vitale b, Rebecca Jane Cox c,d,e, Valerio Stanzani b, Simona Piccirella b, Emanuele Montomoli a,b,⁎ a
Department of Molecular and Developmental Medicine, University of Siena, Via Aldo Moro, 53100 Siena, Italy VisMederi srl, Enterprise in Life Science, Via Fiorentina 1, 53100 Siena, Italy The Influenza Centre, Department of Clinical Sciences, University of Bergen, Bergen, Norway d Jebsen Centre for Influenza Vaccine Research, University of Bergen, Bergen, Norway e Department of Research and Development, Haukeland University Hospital, Bergen, Norway b c
a r t i c l e
i n f o
Article history: Received 28 January 2015 Accepted 15 April 2015 Available online 22 April 2015 Keywords: SRH assay Validation parameters.
a b s t r a c t The Single Radial Haemolysis (SRH) assay is a serological method widely used for measuring antibodies against influenza viruses. Despite the broad application and recommendation by licensing authorities, the SRH assay has not been standardized. The aim of this study is to demonstrate how the SRH assay satisfies validation parameters of regulatory agencies in terms of specificity, precision, repeatability, intermediate precision, linearity, accuracy and robustness. This study shows that the SRH is a rapid, simple, reliable and reproducible assay, which requires only small volumes of serum samples and can be easily standardized. © 2015 Elsevier B.V. All rights reserved.
1. Introduction The Single Radial Haemolysis assay (SRH) is a serological method based on passive haemolysis of erythrocytes (Schild et al., 1975). The haemolysis, mediated by complement and induced by the antibody–antigen complex, produces easily identifiable “areas of haemolysis” which are proportionate to the concentration of anti-influenza antibodies present in serum samples (Morley et al., 1995). The assay is rapid, simple, reliable and reproducible. The main advantage is the ability to simultaneously and rapidly test a large number of samples without pre-treatment (excluding complement inactivation) and the requirement for only a small volume of serum samples. These features make the SRH assay a particularly suitable assay for large-scale investigation especially for epidemiological studies (Clarke et al., 1977; Callow and Beare, 1976; Trombetta et al., 2014). Other significant advantages are the small quantities of virus antigen, the capacity of the assay to detect small differences in antibody levels and the ability to differentiate between closely related influenza strains. In addition, the SRH assay provides numerical results, which are unbiased results available after an overnight incubation (Farrohi et al., 1977; Fulton et al., 1984).
⁎ Corresponding author at: Department of Molecular and Developmental Medicine, University of Siena, Via Aldo Moro, 53100 Siena, Italy. Tel.: +39 0577 234134. E-mail address: [email protected] (E. Montomoli). 1 The authors contributed equally to this work.
http://dx.doi.org/10.1016/j.jim.2015.04.009 0022-1759/© 2015 Elsevier B.V. All rights reserved.
Following natural infection or vaccination, antibodies detected by SRH assay are mainly IgG antibodies. These antibodies are detected in serum samples collected 3–6 weeks after infection or vaccination. In the case of natural infection, the SRH assay is able to detect antibodies up to 12 months with only a small decrease in the titer (Schild et al., 1975). After the appearance of zoonotic avian influenza viruses and ongoing avian outbreaks, the SRH assay has been widely used because of the relatively poor sensitivity of the Haemagglutination Inhibition assay (HI) for detection of antibodies against avian influenza viruses (Rowe et al., 1999; Kayali et al., 2008). Another important advantage of the SRH in the case of avian influenza viruses is its safety because, unlike the Virus Neutralization (VN) assay which requires live influenza viruses, it can be performed with inactivated virus and consequently in BSL 2 containment (Wood et al., 2001). The SRH assay is a technique used to detect antibodies not only against influenza A and B viruses but also against numerous other viruses such as Rubella, Parainfluenza virus, Dengue and Japanese Encephalitis viruses (Schild et al., 1975; Chan et al., 1985; Russell et al., 1978). The SRH assay, together with HI assay, is officially recognized by the European Medicines Agency (EMA) for evaluation of the immunogenicity of a new influenza vaccine and to license annual influenza vaccines (EMA, 1997). Currently, a standardized validation of the SRH assay is not available despite the wide spread use of the assay. Therefore, the aim of this study is to describe the SRH assay on the basis of the validation parameters included in the analytical procedures such as specificity, precision, repeatability, intermediate precision, linearity, accuracy and robustness (EMA, 1995).
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2. Material and methods 2.1. Virus strains The virus antigens were the seasonal egg-derived influenza strains from NIBSC: A/California/7/2009 (H1N1, 10/122), A/Perth/16/2009 (H3N2, 11/106) and B/Brisbane/60/2008 (B, 12/200). 2.2. Serum samples The human serum samples belong to the laboratory serum bank and are from adults aged 17–59 years. Initially, a total of 50 serum samples were analyzed by the HI and VN assays and divided into groups of high positive (HP), low positive (LP) and negative (NP). Serum samples have been defined as high positive when showing a titer ≥ 160 for HI and VN assays and low positive when showing a titer between 40 and 159 for both assays. Negative samples are conventionally expressed as 5, half the lowest detection threshold for both assays. Sera were pooled in the three groups to allow evaluation of the validation parameters. All serum samples were heat-inactivated at 56 °C for 30 min in a water bath before testing in the SRH assay. Influenza sheep antisera provided by NIBSC were used as positive controls: A/California/7/2009 (H1N1, 09/152), A/Perth/16/2009 (H3N2, 09/270) and B/Brisbane/60/ 2008 (B, 11/136). Influenza sheep antisera provided by NIBSC and CBER were used as heterologous controls: A/California/7/2009 (H1N1, 09/152), A/Brisbane/10/2007 (H3N2, 08/246) and A/Indonesia/05/ 2005 (H5N1, S7854). Human serum without IgA, IgM, IgG was used as negative control (Sigma-Aldrich-S5393). 2.3. SRH assay Fresh turkey erythrocytes were centrifuged two times and washed with phosphatebuffersaline(PBS).Dilutedvirusantigen wasaddedtotheerythrocyte suspension at a concentration of 2000 haemagglutinin units (HAU) per millilitres (ml). The erythrocyte–antigen suspension was incubated at 4 °C for 20 min in order to allow the adsorption of virus antigen to the erythrocytes.Asolutionofchromiumchloride(CrCl3)2,5mMwasaddedtoerythrocytes–virus antigen suspension and incubated at room temperature for 10 min, to increase the binding affinity between the erythrocytes and the virus antigen. The suspension was carefully mixed once and then centrifuged. The supernatant was removed, PBS added to the pellet which was carefully re-suspended. A stock solution was prepared of 1.5% agarose in PBS containing 0.1% sodium azide and 0.5% low gelling agarose (to decrease the gelling and melting temperatures). The agarose stock solution was kept at 45 °C in a water bath. Each SRH plate consisted of erythrocyte–virus antigen suspension and guinea pig complement in the agarose mixture. The final suspension was vigorouslyshakenandthenevenlyspreadontoeachplate.Plateswereincubated at room temperature for 30 min and then stored at 4 °C for 30–90 min in order to set the agarose. Holes were punctured in each plate with a calibrated punch and 6 μl of serum samples and controls was added into each hole. The plates were stored in a humid box and incubated at 4 °C for 16– 18 h in the dark. After overnight incubation, the plates were incubated in a water bath at 37 °C for 90 min and then the diameters of haemolysis areas were read in millimeters (mm) with a calibrating viewer. 3. Results
The SRH specificity was evaluated as shown in Table 1. For each strain, the specificity of SRH assay was established through the titration of 5 replicates of each human pooled sera and heterologous hyperimmune sera in 5 different plates. The SRH assay was shown to be highly specific (Fig. 1). All serum samples containing antibodies to the homologous H1N1, H3N2 and B strains had high positive results (include mm2 and standard error) whereas heterologous strains had very small haemolysis areas due to a minimum cross-reactivity. The coefficient of variation (CV) of H1N1, H5N1 and H3N2 Brisbane was 5.14%, 15.06% and 13.15% for H1N1 California strain, 4.28% (H3N2 Perth), 13.15%(H1N1)and 12.53% (H5N1)forH3N2Perthstrain and 4.28%(B/Brisbane), 12.53% (H5N1) and 11.28% (H1N1) for B Brisbane strain.
3.2. Precision “The precision of an analytical procedure expresses the closeness of agreement (degree of scatter) between a series of measurements obtained from multiple sampling of the same homogeneous sample under the prescribed conditions” (EMA, 1995). The precision of an analytical procedure is generally defined as the standard deviation or relative standard deviation (coefficient of variation) of a series of measurements (EMA, 1995; United States Pharmacopeia, U.S.P., 2008). The standard deviation may be evaluated at 3 levels: repeatability, intermediate precision and reproducibility.
3.2.1. Repeatability Repeatability (sometimes called intra-assay precision) shows the precision of the assay when the test is carried out in a laboratory over a relatively short time period using the same operator and equipment. Repeatability is assessed by evaluating variation of replicates (EMA, 1995; World Organisation for Animal Health, O.I.E., 2013; United States Pharmacopeia, U.S.P., 2008). The ICH guidelines recommend assessing repeatability using or a minimum of 9 determinations covering the specified range for the procedure or a minimum of 6 determinations at 100% of the test concentration (EMA, 1995). High positive, low positive and negative human pooled sera for each strain were used to evaluate the SRH repeatability. Influenza sheep antisera as positive control and human serum minus IgA, IgM, and IgG as negative control were included on each plate. The same operator tested all serum samples once on six different plates on the same day. The CV of the HP human pooled sera was 4.88% (H1N1), 5.09% (H3N2), and 5.09% (B), the CV of the LP human pooled sera was 6.89% (H1N1), 8.75% (H3N2) and 8.47% (B) and that of the NP human pooled sera was 0% for all strains (Fig. 2).
Table 1 Serum samples and controls used to evaluate specificity parameter. Strain: Human pooled sera: Hyperimmune sera: Positive control: Negative control: Strain: Human pooled sera: Hyperimmune sera:
3.1. Specificity “Specificity is the ability to assess unequivocally the analyte in the presence of components which may be expected to be present” (EMA, 1995). The specificity detects the ability of the assay to differentiate between similar analytes and in particular to differentiate the target analyte from non target analytes (World Organisation for Animal Health, O.I.E., 2013).
Positive control: Negative control: Strain: Human pooled sera: Hyperimmune sera: Positive control: Negative control:
A/California/7/2009 H1N1 A/California/7/2009 H1N1 (homologous) A/Indonesia/05/2005 H5N1 (heterologous) A/Brisbane/10/2007 H3N2 (heterologous) A/California/7/2009 H1N1 Human serum without IgA, IgM, IgG A/Perth/16/2009 H3N2 A/Perth/16/2009 H3N2 (homologous) A/California/7/2009 H1N1 (heterologous) A/Indonesia/05/2005 H5N1 (heterologous) A/Perth/16/2009 H3N2 Human serum without IgA, IgM, IgG B/Brisbane/60/2008 B/Brisbane/60/2008 (homologous) A/Indonesia/05/2005H5N1 (heterologous) A/California/7/2009 H1N1 (heterologous) B/Brisbane/60/2008 (B) Human serum without IgA, IgM, IgG
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Fig. 1. Specificity for A/H1N1/California, A/H3N2/Perth and B/ Brisbane strains.
The EMA guidelines have not established the precise repeatability parameters regarding the upper and lower limits for the SRH assay. The FDA guidelines have defined the repeatability of the assay “The precision determined at each concentration level should not exceed 15% of the coefficient of variation (CV) except for the lower limit of quantification (LLOQ), where it should not exceed 20% of the CV” (Food and Drug Administration, 2001). If the FDA guidelines were applied, the assay fulfilled all parameters.
3.2.2. Intermediate precision The purpose of intermediate precision is to prove the capacity of the assay to provide reproducible results when random events occur. Variations could include operators, equipment, different days and reagents. The intermediate precision was evaluated by performing two different assays by different operators using different analysis sessions in the same laboratory. In order to evaluate intermediate precision of the SRH assay, HP, LP and NP human pooled sera for each strain were tested in 3 plates by two operators on different days. Positive (influenza sheep antisera) and negative controls (human serum minus IgA, IgM, IgG) were included on each plate. The CV for each HP, LP and NP human pooled sera was very low (2.13%–4.45%) and no variation was found in half of the results (Fig. 3). As for Repeatability, if the FDA guidelines are applied, the SRH assay results fulfill the guidelines (Food and Drug Administration, 2001). Importantly, the mean of CV between the three human pooled sera pool is 0.71% for the H1N1 strain, 1.48% for the H3N2 strain and 0% for the B strain, highlighting the great degree of intermediate precision of SRH assay.
Haemolysis Area
H1N1 California
CV
3.3. Linearity “The linearity of an analytical procedure is its ability (within a given range) to obtain test results which are directly proportional to the concentration (amount) of analyte in the sample” (EMA, 1995). The term “linearity” means the linearity of the relationship between the concentration and the assay measurement (United States Pharmacopeia, U.S.P., 2008). Such definition may not be applied to some immunoassays where the relationship is not linear. Therefore, the FDA guidelines use the term “Calibration/Standard Curve” instead of linearity (Food and Drug Administration, 2001; McPolin, 2009). This parameter needs to be demonstrated directly for the tested analyte and to be evaluated by visual examination of a plot of signals as a function of analyte concentration (EMA, 1995). The aim of linearity is to provide a model, linear or not, that is able to illustrate the relationship between concentration and response of the analyte (United States Pharmacopeia, U.S.P., 2008). In the case of a linear relationship, the results should be estimated by appropriate statistical methods. Sometimes, in order to achieve linearity between the assays and the sample concentrations, it could be necessary to modify test data with a mathematical transformation prior to the regression analysis. The classical acceptance criteria for linearity require that the correlation coefficient of the linear regression line is close to 1 and that the y-intercept should not differ significantly from zero. In the case of a significant non zero intercept, it is necessary to demonstrate that it does not have consequences on the accuracy of the assay (World Organisation for Animal Health, O.I.E., 2013).
H3N2 Perth
B Brisbane
HP
LP
NP
HP
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NP
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LP: Low Positive
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Human Poole Sera; NP: Negative Human Pooled Sera;
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4.88%
6.89%
0.00%
5.09%
8.75%
0.00%
5.09%
8.47%
0.00%
Human Pooled Sera;
Fig. 2. Repeatability of the SRH assay. Results obtained by testing 6 determinations at 100% of the test concentration for each strain.
C.M. Trombetta et al. / Journal of Immunological Methods 422 (2015) 95–101
Haemolysis Area
98
CV
HP
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NP
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H3N2 Perth LP
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B Brisbane LP
NP
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2.13%
0.00%
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4.45%
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2.19%
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Fig. 3. The intermediate precision determined in two different analysis sessions which were differentiated by operator and day. HP: High Positive Human Pooled Sera; LP: Low Positive Human Pooled Sera; NP: Negative Human Pooled Sera; CV: coefficient of variation.
The evaluation of the SRH linearity has been established on the titres of 5 dilutions (as recommended by ICH guidelines EMA, 1995) of all HP human pooled sera. The starting point is the undiluted sample followed by serial two fold dilutions in PBS (Fig. 4). The calculated coefficient of correlation is an absolute value number close to 1 for each strain (0.94 for H1N1 California and B Brisbane and 0.96 for H3N2 Perth) and would therefore be acceptable. 3.4. Accuracy “The accuracy of an analytical procedure expresses the closeness of agreement between the value which is accepted either as a conventional true value or an accepted reference value and the value found” (EMA, 1995). Several methods to determine the accuracy are available such as the assessment of the percent recovery throughout the range of the assay or the evaluation of linearity of the relationship between estimated and actual concentrations. The statistically preferred criterion is for the confidence interval of the slope to include 1.0 or alternatively that the slope itself is close to 1.0 (EMA, 1995; United States Pharmacopeia, U.S.P., 2008). Accuracy may be evaluated once precision, linearity and specificity have been established. According to ICH guidelines, 9 determinations of HP human pooled sera have been performed (3 concentrations of pooled sera, undiluted—1:4–1:16, repeated three times). The CV for undiluted sera was 5.37% for the H1N1 strain, 6.88% for the H3N2 strain and 5.37% for B the strain. The diluted sera gave values of 0% for the H3N2 strain and 0–7.95% for the H1N1 and B strains at 1:4 and 1:16 dilution of sera. These results fully meet the FDA guidelines acceptance criteria, for which “The mean value should be within 15% of the actual value except for the lower limit of quantification (LLOQ), where it should not exceed 20% of the CV” (Food and Drug Administration, 2001). Accuracy can also be described as the percentage recovery (EMA, 1995). Table 2 shows the percentage of recovery of the H1N1, H3N2 and B HP human pooled sera and reveals that in the case of 1:4 dilution, the percentage of recovery was 41.73%, 43.05% and 39.82% from the undiluted sera pool (100%) while in case of 1:16 dilution the percentage of recovery from the undiluted sample was 13.63%, 14.06% and 13.63%. 3.5. Robustness “The robustness of an analytical procedure is a measure of its capacity to remain unaffected by small, but deliberate variations in method parameters and provides an indication of its reliability during normal usage” (EMA, 1995). The robustness gives an indication of the assay reliability when events could occur during testing in a single laboratory. The parameters evaluated in order to assess the SRH assay robustness were the virus antigen concentration(Ag), the incubation time(IncTm) at + 4 °C, the incubation time + 37 °C, the amount of serum sample and the different batches of turkey erythrocytes(RBC).
For each parameter and strain, the HP human pooled sera were tested 6 times by one operator on the same day (Table 3). In order to evaluate the relationship between the virus antigen concentration and the haemolysis areas, the HP human pooled sera for each strain were tested 6 times by one operator during the same analysis session. The initial virus antigen concentration of 2000 haemagglutinin units per ml was used as the standard condition, robustness parameter evaluated the variation of virus antigen concentrations (± 500 haemagglutinin units per ml). The parameter “incubation time at + 4 °C” aimed to evaluate the differences in the haemolysis area after a shorter (14 h) or longer (18 h) overnight incubation than the standard condition (16 h). The SRH assay has two options in order to allow the diffusion of the analyzed serum samples. The first option is an overnight incubation of plates at 37 °C, the second is an overnight incubation at 4 °C followed by a supplementary incubation at 37 °C for 2–3 h (Chan et al., 1985). The standard procedure in this study was an incubation at 37 °C for 90 min. The conventional amount of serum samples seeded in the plates is 6 μl. Small variations in the amounts of serum samples were evaluated (range 5.5 μl to 6.5 μl). Diverse batches of turkey erythrocytes were evaluated to test for differences in haemolysis area. As shown in Fig. 5, the SRH assay was not affected by variations of incubation times, the amount of serum samples, the virus antigen concentration and different batches of turkey erythrocytes providing further evidence of its reliability. 3.6. Discussion The term “validate a method” stands for “evaluate the fitness of the methods” (World Organisation for Animal Health, O.I.E., 2013). It is important to emphasize that the main purpose of a validation of an analytical procedure is to provide evidence that the method is suitable for its intended purpose (EMA, 1995). The approach outlined in this work aimed to estimate how the SRH assay is able to fulfill the required criteria of regulatory agencies and to validate the assay which is one of the methods officially recognized to evaluate the immunogenicity of a new influenza vaccine and to license the vaccine. The validation experiments have been performed with high, low and negative human pooled serum samples, influenza sheep antisera provided by NIBSC and CBER and the seasonal egg-derived influenza strains A/California/7/2009 (H1N1), A/Perth/16/2009 (H3N2) and B/ Brisbane/60/2008. The parameters considered were: Specificity, Precision (that includes Repeatability and Intermediate Precision), Linearity, Accuracy and Robustness. All these parameters evaluate how the assay can guarantee the reliability in different circumstances. Specificity is a crucial parameter in order to license a vaccine because it shows how the measured antibody response is specific for the strain of interest. The aim of this parameter is to differentiate between the
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Fig. 4. A. Linearity parameter—tendency line for A/H1N1/California strain (human pooled sera homologous for H1N1 California strain). B. Linearity parameter—tendency line for A/H3N2/ Perth strain (human pooled sera homologous for H3N2 Perth strain). C. Linearity parameter—tendency line for B/ Brisbane strain (human pooled sera homologous for B Brisbane strain).
Table 2 Percentage of recovery. H1N1 California
H3N2 Perth
B Brisbane
Undiluted
1:4
1:16
Undiluted
1:4
1:16
Undiluted
1:4
1:16
100%
41.73%
13.63%
100%
43.05%
14.06%
100%
39.82%
13.63%
100
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Table 3 Robustness parameters. HP
Standard conditions
Virus concentration (HAU/ml) Incubation time at +4 °C Incubation time at +37 °C Amount of sera Different batches of erythrocytes
1500 14 h 60′ 5.5 μl X
2000 16 h 90′ 6 μl Y
2500 18 h 120′ 6.5 μl Z
target analyte from similar non target analytes. The results demonstrate the high degree of specificity of the SRH assay, which is able to discriminate between homologous and closely related influenza strains. We have shown that the homologous strains always give high positive
results while heterologous strains result in a barely detectable haemolysis. These results were confirmed using positive and negative controls for each strain. The evaluation of specificity could be more deeply investigated adding heterologous strains from the same subtype (e.g. A/Perth/16/ 2009 H3N2 vs A/Switzerland/9715293/2013 H3N2). The SRH assay was a very repeatable method as observed by the evaluation of repeatability (intra assay) and intermediate precision. The repeatability highlights the precision of the assay to evaluate the variation in results when the test is carried out in a laboratory over a relatively short time period using the same operator and equipment. The EMA guidelines have not established the precise repeatability parameters while the FDA guidelines give an indication of the
Fig. 5. A. Robustness parameter for A/H1N1/California strain. B. Robustness parameter for A/H3N2/Perthstrain. C. Robustness parameter for B/ Brisbane strain.
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repeatability of the assay. If these guidelines were applied, the results of HP, LP and NP human pooled sera for each strain fulfilled all parameters. The CV for all the serum samples showed the high precision of the assay under the same operating conditions. Beyond the repeatability of the SRH assay, this work estimates the effects of random events such as different days and different equipment with the intermediate precision parameter. This parameter was evaluated by performing two different assays by different operators on different analysis sessions in the same laboratory. The CV for each HP, LP and NP human pooled sera was very low and no variation was found in half of the results. The data highlight the great degree of intermediate precision of SRH assay and the capacity to guarantee the same results in different analysis sessions. The precision also includes reproducibility parameter that entails the ability of the assay to provide the same results when performed in different laboratories which has not been evaluated but should be analyzed in further work. The robustness parameter gives an indication of the assay reliability when minor variations occur during testing in a single laboratory. The evaluated variations from the standard procedure were incubation time at different temperatures, initial concentration of the virus, the amount of serum sample and different batches of erythrocytes. We found complete compliance between these variations and the results obtained using standard conditions, highlighting the capacity of the assay to be unaffected by small variations and underlining the high reliability of the assay. Two parameters further estimated are the linearity and accuracy of the assay. The aim of linearity is to illustrate the relationship between concentration and response of the analyte. While the accuracy, evaluated once precision, linearity and specificity have been established, expresses the agreement between the conventional or accepted true value and those found. Our results support the concept of the SRH as a linear and accurate technique in addition to the other features shown above. The stringent demonstration of the SRH assay to meet all regulatory guidelines further increases the advantage of this assay as an easy technique, relatively cheap and allowing the simultaneous testing of a large number of samples without any pre-treatment. In conclusion, this study shows for the first time that the SRH assay is a technique that has the advantage of rapidity and simplicity (Schild et al., 1975) and is not affected by the subjectivity of the operator (Fulton et al., 1984). Furthermore we have confirmed that the SRH is a robust and specific technique for detection of strain specific antibodies against influenza virus. Author contribution All authors contributed to review and revise the manuscript. Conflicts of interest The authors declare no conflict of interest.
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Acknowledgment The work leading to these results has received support from the Innovative Medicines Initiative Joint Undertaking under grant agreement n° 115672, resources of which are composed of financial contribution from the European Union's Seventh Framework Programme (FP7/ 2007-2013) and EFPIA companies’ in kind contribution. References Callow, K.A., Beare, A.S., 1976. Measurement of antibody to influenza virus neuraminidase by single radial hemolysis in agarose gels. Infect. Immun. 13 (1), 1. Chan, Y.C., Tan, H.C., Tan, S.H., Balachandran, K., 1985. The use of the single radial haemolysis technique in the serological diagnosis of dengue and Japanese encephalitis virus infections. Bull. World Health Organ. 63 (6), 1043. Clarke, M., Boustred, J., Seagroatt, V., Schild, G.C., 1977. The use of single-radialhaemolysis for rubella antibody studies. J. Hyg. (Lond.) 79 (3), 355. EMA, 1995. Q2(R1) Validation of Analytical Procedures: Text and Methodology (CPMP/ ICH/381/95). EMA, 1997. Note for Guidance on Harmonisation of Requirements for Influenza Vaccines (CPMP/BWP/214/96). Farrohi, K., Farrohi, F.K., Noble, G.R., Kaye, H.S., Kendal, A.P., 1977. Evaluation of the single radial hemolysis test for measuring hemagglutinin- and neuraminidase-specific antibodies to H3N2 influenza strains and antibodies to influenza B. J. Clin. Microbiol. 5 (3), 353. Food and Drug Administration, 2001. Bioanalytical Method Validation. p. 4. Fulton, R.E., DiNinno, V.L., Frank, R.I., Fildes, J., Turner, I.J., 1984. Single radial hemolysis test for quantitation of complement-fixing antibodies to non-hemagglutinating viruses. J. Clin. Microbiol. 20 (2), 248. Kayali, G., Setterquist, S.F., Capuano, A.W., Myers, K.P., Gill, J.S., Gray, G.C., 2008. Testing human sera for antibodies against avian influenza viruses: horse RBC hemagglutination inhibition vs. microneutralization assays. J. Clin. Virol. 43 (1), 73. McPolin, O., 2009. Validation of Analytical Methods for Pharmaceutical Analysis. Mourne Training Services. Morley, P.S., Hanson, L.K., Bogdan, J.R., Townsend, H.G., Appleton, J.A., Haines, D.M., 1995. The relationship between single radial hemolysis, hemagglutination inhibition, and virus neutralization assays used to detect antibodies specific for equine influenza viruses. Vet. Microbiol. 45 (1), 81. Rowe, T., Abernathy, R.A., Hu-Primmer, J., Thompson, W.W., Lu, X., Lim, W., Fukuda, K., Cox, N.J., Katz, J.M., 1999. Detection of antibody to avian influenza A (H5N1) virus in human serum by using a combination of serologic assays. J. Clin. Microbiol. 37 (4), 937. Russell, S.M., Benjamin, S.R., Briggs, M., Jenkins, M., Mortimer, P.P., Payne, S.B., 1978. Evaluation of the single radial haemolysis (SRH) technique for rubella antibody measurement. J. Clin. Pathol. 31 (6), 521. Schild, G.C., Pereira, M.S., Chakraverty, P., 1975. Single-radial-hemolysis: a new method for the assay of antibody to influenza haemagglutinin. Applications for diagnosis and seroepidemiologic surveillance of influenza. Bull. World Health Organ. 52 (1), 43. Trombetta, C.M., Perini, D., Mather, S., Temperon, N., Montomoli, E., 2014. Overview of serological techniques for influenza vaccine evaluation: past, present and future. Vaccines 2 (4), 707. United States Pharmacopeia, U.S.P., 2008. Validation of Compendial Procedures. p. 1. Wood, J.M., Melzack, D., Newman, R.W., Major, D.L., Zambon, M., Nicholson, K.G., Podda, A., 2001. A single radial haemolysis assay for antibodyto H5 haemagglutinin. Int. Congr. Ser. 1219, 761. World Organisation for Animal Health, O.I.E., 2013. Principles and methods of validation of diagnostic assays for infectious diseases. Manual of Diagnostic Tests and Vaccines for Terrestrial Animals vol. 1.
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Journal of Immunological Methods journal homepage: www.elsevier.com/locate/jim
Research paper
Validation of Single Radial Haemolysis assay: A reliable method to measure antibodies against influenza viruses Claudia Maria Trombetta a,1, Daniele Perini b,1, Licia Vitale b, Rebecca Jane Cox c,d,e, Valerio Stanzani b, Simona Piccirella b, Emanuele Montomoli a,b,⁎ a
Department of Molecular and Developmental Medicine, University of Siena, Via Aldo Moro, 53100 Siena, Italy VisMederi srl, Enterprise in Life Science, Via Fiorentina 1, 53100 Siena, Italy The Influenza Centre, Department of Clinical Sciences, University of Bergen, Bergen, Norway d Jebsen Centre for Influenza Vaccine Research, University of Bergen, Bergen, Norway e Department of Research and Development, Haukeland University Hospital, Bergen, Norway b c
a r t i c l e
i n f o
Article history: Received 28 January 2015 Accepted 15 April 2015 Available online 22 April 2015 Keywords: SRH assay Validation parameters.
a b s t r a c t The Single Radial Haemolysis (SRH) assay is a serological method widely used for measuring antibodies against influenza viruses. Despite the broad application and recommendation by licensing authorities, the SRH assay has not been standardized. The aim of this study is to demonstrate how the SRH assay satisfies validation parameters of regulatory agencies in terms of specificity, precision, repeatability, intermediate precision, linearity, accuracy and robustness. This study shows that the SRH is a rapid, simple, reliable and reproducible assay, which requires only small volumes of serum samples and can be easily standardized. © 2015 Elsevier B.V. All rights reserved.
1. Introduction The Single Radial Haemolysis assay (SRH) is a serological method based on passive haemolysis of erythrocytes (Schild et al., 1975). The haemolysis, mediated by complement and induced by the antibody–antigen complex, produces easily identifiable “areas of haemolysis” which are proportionate to the concentration of anti-influenza antibodies present in serum samples (Morley et al., 1995). The assay is rapid, simple, reliable and reproducible. The main advantage is the ability to simultaneously and rapidly test a large number of samples without pre-treatment (excluding complement inactivation) and the requirement for only a small volume of serum samples. These features make the SRH assay a particularly suitable assay for large-scale investigation especially for epidemiological studies (Clarke et al., 1977; Callow and Beare, 1976; Trombetta et al., 2014). Other significant advantages are the small quantities of virus antigen, the capacity of the assay to detect small differences in antibody levels and the ability to differentiate between closely related influenza strains. In addition, the SRH assay provides numerical results, which are unbiased results available after an overnight incubation (Farrohi et al., 1977; Fulton et al., 1984).
⁎ Corresponding author at: Department of Molecular and Developmental Medicine, University of Siena, Via Aldo Moro, 53100 Siena, Italy. Tel.: +39 0577 234134. E-mail address: [email protected] (E. Montomoli). 1 The authors contributed equally to this work.
http://dx.doi.org/10.1016/j.jim.2015.04.009 0022-1759/© 2015 Elsevier B.V. All rights reserved.
Following natural infection or vaccination, antibodies detected by SRH assay are mainly IgG antibodies. These antibodies are detected in serum samples collected 3–6 weeks after infection or vaccination. In the case of natural infection, the SRH assay is able to detect antibodies up to 12 months with only a small decrease in the titer (Schild et al., 1975). After the appearance of zoonotic avian influenza viruses and ongoing avian outbreaks, the SRH assay has been widely used because of the relatively poor sensitivity of the Haemagglutination Inhibition assay (HI) for detection of antibodies against avian influenza viruses (Rowe et al., 1999; Kayali et al., 2008). Another important advantage of the SRH in the case of avian influenza viruses is its safety because, unlike the Virus Neutralization (VN) assay which requires live influenza viruses, it can be performed with inactivated virus and consequently in BSL 2 containment (Wood et al., 2001). The SRH assay is a technique used to detect antibodies not only against influenza A and B viruses but also against numerous other viruses such as Rubella, Parainfluenza virus, Dengue and Japanese Encephalitis viruses (Schild et al., 1975; Chan et al., 1985; Russell et al., 1978). The SRH assay, together with HI assay, is officially recognized by the European Medicines Agency (EMA) for evaluation of the immunogenicity of a new influenza vaccine and to license annual influenza vaccines (EMA, 1997). Currently, a standardized validation of the SRH assay is not available despite the wide spread use of the assay. Therefore, the aim of this study is to describe the SRH assay on the basis of the validation parameters included in the analytical procedures such as specificity, precision, repeatability, intermediate precision, linearity, accuracy and robustness (EMA, 1995).
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2. Material and methods 2.1. Virus strains The virus antigens were the seasonal egg-derived influenza strains from NIBSC: A/California/7/2009 (H1N1, 10/122), A/Perth/16/2009 (H3N2, 11/106) and B/Brisbane/60/2008 (B, 12/200). 2.2. Serum samples The human serum samples belong to the laboratory serum bank and are from adults aged 17–59 years. Initially, a total of 50 serum samples were analyzed by the HI and VN assays and divided into groups of high positive (HP), low positive (LP) and negative (NP). Serum samples have been defined as high positive when showing a titer ≥ 160 for HI and VN assays and low positive when showing a titer between 40 and 159 for both assays. Negative samples are conventionally expressed as 5, half the lowest detection threshold for both assays. Sera were pooled in the three groups to allow evaluation of the validation parameters. All serum samples were heat-inactivated at 56 °C for 30 min in a water bath before testing in the SRH assay. Influenza sheep antisera provided by NIBSC were used as positive controls: A/California/7/2009 (H1N1, 09/152), A/Perth/16/2009 (H3N2, 09/270) and B/Brisbane/60/ 2008 (B, 11/136). Influenza sheep antisera provided by NIBSC and CBER were used as heterologous controls: A/California/7/2009 (H1N1, 09/152), A/Brisbane/10/2007 (H3N2, 08/246) and A/Indonesia/05/ 2005 (H5N1, S7854). Human serum without IgA, IgM, IgG was used as negative control (Sigma-Aldrich-S5393). 2.3. SRH assay Fresh turkey erythrocytes were centrifuged two times and washed with phosphatebuffersaline(PBS).Dilutedvirusantigen wasaddedtotheerythrocyte suspension at a concentration of 2000 haemagglutinin units (HAU) per millilitres (ml). The erythrocyte–antigen suspension was incubated at 4 °C for 20 min in order to allow the adsorption of virus antigen to the erythrocytes.Asolutionofchromiumchloride(CrCl3)2,5mMwasaddedtoerythrocytes–virus antigen suspension and incubated at room temperature for 10 min, to increase the binding affinity between the erythrocytes and the virus antigen. The suspension was carefully mixed once and then centrifuged. The supernatant was removed, PBS added to the pellet which was carefully re-suspended. A stock solution was prepared of 1.5% agarose in PBS containing 0.1% sodium azide and 0.5% low gelling agarose (to decrease the gelling and melting temperatures). The agarose stock solution was kept at 45 °C in a water bath. Each SRH plate consisted of erythrocyte–virus antigen suspension and guinea pig complement in the agarose mixture. The final suspension was vigorouslyshakenandthenevenlyspreadontoeachplate.Plateswereincubated at room temperature for 30 min and then stored at 4 °C for 30–90 min in order to set the agarose. Holes were punctured in each plate with a calibrated punch and 6 μl of serum samples and controls was added into each hole. The plates were stored in a humid box and incubated at 4 °C for 16– 18 h in the dark. After overnight incubation, the plates were incubated in a water bath at 37 °C for 90 min and then the diameters of haemolysis areas were read in millimeters (mm) with a calibrating viewer. 3. Results
The SRH specificity was evaluated as shown in Table 1. For each strain, the specificity of SRH assay was established through the titration of 5 replicates of each human pooled sera and heterologous hyperimmune sera in 5 different plates. The SRH assay was shown to be highly specific (Fig. 1). All serum samples containing antibodies to the homologous H1N1, H3N2 and B strains had high positive results (include mm2 and standard error) whereas heterologous strains had very small haemolysis areas due to a minimum cross-reactivity. The coefficient of variation (CV) of H1N1, H5N1 and H3N2 Brisbane was 5.14%, 15.06% and 13.15% for H1N1 California strain, 4.28% (H3N2 Perth), 13.15%(H1N1)and 12.53% (H5N1)forH3N2Perthstrain and 4.28%(B/Brisbane), 12.53% (H5N1) and 11.28% (H1N1) for B Brisbane strain.
3.2. Precision “The precision of an analytical procedure expresses the closeness of agreement (degree of scatter) between a series of measurements obtained from multiple sampling of the same homogeneous sample under the prescribed conditions” (EMA, 1995). The precision of an analytical procedure is generally defined as the standard deviation or relative standard deviation (coefficient of variation) of a series of measurements (EMA, 1995; United States Pharmacopeia, U.S.P., 2008). The standard deviation may be evaluated at 3 levels: repeatability, intermediate precision and reproducibility.
3.2.1. Repeatability Repeatability (sometimes called intra-assay precision) shows the precision of the assay when the test is carried out in a laboratory over a relatively short time period using the same operator and equipment. Repeatability is assessed by evaluating variation of replicates (EMA, 1995; World Organisation for Animal Health, O.I.E., 2013; United States Pharmacopeia, U.S.P., 2008). The ICH guidelines recommend assessing repeatability using or a minimum of 9 determinations covering the specified range for the procedure or a minimum of 6 determinations at 100% of the test concentration (EMA, 1995). High positive, low positive and negative human pooled sera for each strain were used to evaluate the SRH repeatability. Influenza sheep antisera as positive control and human serum minus IgA, IgM, and IgG as negative control were included on each plate. The same operator tested all serum samples once on six different plates on the same day. The CV of the HP human pooled sera was 4.88% (H1N1), 5.09% (H3N2), and 5.09% (B), the CV of the LP human pooled sera was 6.89% (H1N1), 8.75% (H3N2) and 8.47% (B) and that of the NP human pooled sera was 0% for all strains (Fig. 2).
Table 1 Serum samples and controls used to evaluate specificity parameter. Strain: Human pooled sera: Hyperimmune sera: Positive control: Negative control: Strain: Human pooled sera: Hyperimmune sera:
3.1. Specificity “Specificity is the ability to assess unequivocally the analyte in the presence of components which may be expected to be present” (EMA, 1995). The specificity detects the ability of the assay to differentiate between similar analytes and in particular to differentiate the target analyte from non target analytes (World Organisation for Animal Health, O.I.E., 2013).
Positive control: Negative control: Strain: Human pooled sera: Hyperimmune sera: Positive control: Negative control:
A/California/7/2009 H1N1 A/California/7/2009 H1N1 (homologous) A/Indonesia/05/2005 H5N1 (heterologous) A/Brisbane/10/2007 H3N2 (heterologous) A/California/7/2009 H1N1 Human serum without IgA, IgM, IgG A/Perth/16/2009 H3N2 A/Perth/16/2009 H3N2 (homologous) A/California/7/2009 H1N1 (heterologous) A/Indonesia/05/2005 H5N1 (heterologous) A/Perth/16/2009 H3N2 Human serum without IgA, IgM, IgG B/Brisbane/60/2008 B/Brisbane/60/2008 (homologous) A/Indonesia/05/2005H5N1 (heterologous) A/California/7/2009 H1N1 (heterologous) B/Brisbane/60/2008 (B) Human serum without IgA, IgM, IgG
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Fig. 1. Specificity for A/H1N1/California, A/H3N2/Perth and B/ Brisbane strains.
The EMA guidelines have not established the precise repeatability parameters regarding the upper and lower limits for the SRH assay. The FDA guidelines have defined the repeatability of the assay “The precision determined at each concentration level should not exceed 15% of the coefficient of variation (CV) except for the lower limit of quantification (LLOQ), where it should not exceed 20% of the CV” (Food and Drug Administration, 2001). If the FDA guidelines were applied, the assay fulfilled all parameters.
3.2.2. Intermediate precision The purpose of intermediate precision is to prove the capacity of the assay to provide reproducible results when random events occur. Variations could include operators, equipment, different days and reagents. The intermediate precision was evaluated by performing two different assays by different operators using different analysis sessions in the same laboratory. In order to evaluate intermediate precision of the SRH assay, HP, LP and NP human pooled sera for each strain were tested in 3 plates by two operators on different days. Positive (influenza sheep antisera) and negative controls (human serum minus IgA, IgM, IgG) were included on each plate. The CV for each HP, LP and NP human pooled sera was very low (2.13%–4.45%) and no variation was found in half of the results (Fig. 3). As for Repeatability, if the FDA guidelines are applied, the SRH assay results fulfill the guidelines (Food and Drug Administration, 2001). Importantly, the mean of CV between the three human pooled sera pool is 0.71% for the H1N1 strain, 1.48% for the H3N2 strain and 0% for the B strain, highlighting the great degree of intermediate precision of SRH assay.
Haemolysis Area
H1N1 California
CV
3.3. Linearity “The linearity of an analytical procedure is its ability (within a given range) to obtain test results which are directly proportional to the concentration (amount) of analyte in the sample” (EMA, 1995). The term “linearity” means the linearity of the relationship between the concentration and the assay measurement (United States Pharmacopeia, U.S.P., 2008). Such definition may not be applied to some immunoassays where the relationship is not linear. Therefore, the FDA guidelines use the term “Calibration/Standard Curve” instead of linearity (Food and Drug Administration, 2001; McPolin, 2009). This parameter needs to be demonstrated directly for the tested analyte and to be evaluated by visual examination of a plot of signals as a function of analyte concentration (EMA, 1995). The aim of linearity is to provide a model, linear or not, that is able to illustrate the relationship between concentration and response of the analyte (United States Pharmacopeia, U.S.P., 2008). In the case of a linear relationship, the results should be estimated by appropriate statistical methods. Sometimes, in order to achieve linearity between the assays and the sample concentrations, it could be necessary to modify test data with a mathematical transformation prior to the regression analysis. The classical acceptance criteria for linearity require that the correlation coefficient of the linear regression line is close to 1 and that the y-intercept should not differ significantly from zero. In the case of a significant non zero intercept, it is necessary to demonstrate that it does not have consequences on the accuracy of the assay (World Organisation for Animal Health, O.I.E., 2013).
H3N2 Perth
B Brisbane
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LP
NP
HP
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LP: Low Positive
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Human Poole Sera; NP: Negative Human Pooled Sera;
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33.17
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33.17
3.99
4.88%
6.89%
0.00%
5.09%
8.75%
0.00%
5.09%
8.47%
0.00%
Human Pooled Sera;
Fig. 2. Repeatability of the SRH assay. Results obtained by testing 6 determinations at 100% of the test concentration for each strain.
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Haemolysis Area
98
CV
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2.13%
0.00%
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Fig. 3. The intermediate precision determined in two different analysis sessions which were differentiated by operator and day. HP: High Positive Human Pooled Sera; LP: Low Positive Human Pooled Sera; NP: Negative Human Pooled Sera; CV: coefficient of variation.
The evaluation of the SRH linearity has been established on the titres of 5 dilutions (as recommended by ICH guidelines EMA, 1995) of all HP human pooled sera. The starting point is the undiluted sample followed by serial two fold dilutions in PBS (Fig. 4). The calculated coefficient of correlation is an absolute value number close to 1 for each strain (0.94 for H1N1 California and B Brisbane and 0.96 for H3N2 Perth) and would therefore be acceptable. 3.4. Accuracy “The accuracy of an analytical procedure expresses the closeness of agreement between the value which is accepted either as a conventional true value or an accepted reference value and the value found” (EMA, 1995). Several methods to determine the accuracy are available such as the assessment of the percent recovery throughout the range of the assay or the evaluation of linearity of the relationship between estimated and actual concentrations. The statistically preferred criterion is for the confidence interval of the slope to include 1.0 or alternatively that the slope itself is close to 1.0 (EMA, 1995; United States Pharmacopeia, U.S.P., 2008). Accuracy may be evaluated once precision, linearity and specificity have been established. According to ICH guidelines, 9 determinations of HP human pooled sera have been performed (3 concentrations of pooled sera, undiluted—1:4–1:16, repeated three times). The CV for undiluted sera was 5.37% for the H1N1 strain, 6.88% for the H3N2 strain and 5.37% for B the strain. The diluted sera gave values of 0% for the H3N2 strain and 0–7.95% for the H1N1 and B strains at 1:4 and 1:16 dilution of sera. These results fully meet the FDA guidelines acceptance criteria, for which “The mean value should be within 15% of the actual value except for the lower limit of quantification (LLOQ), where it should not exceed 20% of the CV” (Food and Drug Administration, 2001). Accuracy can also be described as the percentage recovery (EMA, 1995). Table 2 shows the percentage of recovery of the H1N1, H3N2 and B HP human pooled sera and reveals that in the case of 1:4 dilution, the percentage of recovery was 41.73%, 43.05% and 39.82% from the undiluted sera pool (100%) while in case of 1:16 dilution the percentage of recovery from the undiluted sample was 13.63%, 14.06% and 13.63%. 3.5. Robustness “The robustness of an analytical procedure is a measure of its capacity to remain unaffected by small, but deliberate variations in method parameters and provides an indication of its reliability during normal usage” (EMA, 1995). The robustness gives an indication of the assay reliability when events could occur during testing in a single laboratory. The parameters evaluated in order to assess the SRH assay robustness were the virus antigen concentration(Ag), the incubation time(IncTm) at + 4 °C, the incubation time + 37 °C, the amount of serum sample and the different batches of turkey erythrocytes(RBC).
For each parameter and strain, the HP human pooled sera were tested 6 times by one operator on the same day (Table 3). In order to evaluate the relationship between the virus antigen concentration and the haemolysis areas, the HP human pooled sera for each strain were tested 6 times by one operator during the same analysis session. The initial virus antigen concentration of 2000 haemagglutinin units per ml was used as the standard condition, robustness parameter evaluated the variation of virus antigen concentrations (± 500 haemagglutinin units per ml). The parameter “incubation time at + 4 °C” aimed to evaluate the differences in the haemolysis area after a shorter (14 h) or longer (18 h) overnight incubation than the standard condition (16 h). The SRH assay has two options in order to allow the diffusion of the analyzed serum samples. The first option is an overnight incubation of plates at 37 °C, the second is an overnight incubation at 4 °C followed by a supplementary incubation at 37 °C for 2–3 h (Chan et al., 1985). The standard procedure in this study was an incubation at 37 °C for 90 min. The conventional amount of serum samples seeded in the plates is 6 μl. Small variations in the amounts of serum samples were evaluated (range 5.5 μl to 6.5 μl). Diverse batches of turkey erythrocytes were evaluated to test for differences in haemolysis area. As shown in Fig. 5, the SRH assay was not affected by variations of incubation times, the amount of serum samples, the virus antigen concentration and different batches of turkey erythrocytes providing further evidence of its reliability. 3.6. Discussion The term “validate a method” stands for “evaluate the fitness of the methods” (World Organisation for Animal Health, O.I.E., 2013). It is important to emphasize that the main purpose of a validation of an analytical procedure is to provide evidence that the method is suitable for its intended purpose (EMA, 1995). The approach outlined in this work aimed to estimate how the SRH assay is able to fulfill the required criteria of regulatory agencies and to validate the assay which is one of the methods officially recognized to evaluate the immunogenicity of a new influenza vaccine and to license the vaccine. The validation experiments have been performed with high, low and negative human pooled serum samples, influenza sheep antisera provided by NIBSC and CBER and the seasonal egg-derived influenza strains A/California/7/2009 (H1N1), A/Perth/16/2009 (H3N2) and B/ Brisbane/60/2008. The parameters considered were: Specificity, Precision (that includes Repeatability and Intermediate Precision), Linearity, Accuracy and Robustness. All these parameters evaluate how the assay can guarantee the reliability in different circumstances. Specificity is a crucial parameter in order to license a vaccine because it shows how the measured antibody response is specific for the strain of interest. The aim of this parameter is to differentiate between the
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Fig. 4. A. Linearity parameter—tendency line for A/H1N1/California strain (human pooled sera homologous for H1N1 California strain). B. Linearity parameter—tendency line for A/H3N2/ Perth strain (human pooled sera homologous for H3N2 Perth strain). C. Linearity parameter—tendency line for B/ Brisbane strain (human pooled sera homologous for B Brisbane strain).
Table 2 Percentage of recovery. H1N1 California
H3N2 Perth
B Brisbane
Undiluted
1:4
1:16
Undiluted
1:4
1:16
Undiluted
1:4
1:16
100%
41.73%
13.63%
100%
43.05%
14.06%
100%
39.82%
13.63%
100
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Table 3 Robustness parameters. HP
Standard conditions
Virus concentration (HAU/ml) Incubation time at +4 °C Incubation time at +37 °C Amount of sera Different batches of erythrocytes
1500 14 h 60′ 5.5 μl X
2000 16 h 90′ 6 μl Y
2500 18 h 120′ 6.5 μl Z
target analyte from similar non target analytes. The results demonstrate the high degree of specificity of the SRH assay, which is able to discriminate between homologous and closely related influenza strains. We have shown that the homologous strains always give high positive
results while heterologous strains result in a barely detectable haemolysis. These results were confirmed using positive and negative controls for each strain. The evaluation of specificity could be more deeply investigated adding heterologous strains from the same subtype (e.g. A/Perth/16/ 2009 H3N2 vs A/Switzerland/9715293/2013 H3N2). The SRH assay was a very repeatable method as observed by the evaluation of repeatability (intra assay) and intermediate precision. The repeatability highlights the precision of the assay to evaluate the variation in results when the test is carried out in a laboratory over a relatively short time period using the same operator and equipment. The EMA guidelines have not established the precise repeatability parameters while the FDA guidelines give an indication of the
Fig. 5. A. Robustness parameter for A/H1N1/California strain. B. Robustness parameter for A/H3N2/Perthstrain. C. Robustness parameter for B/ Brisbane strain.
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repeatability of the assay. If these guidelines were applied, the results of HP, LP and NP human pooled sera for each strain fulfilled all parameters. The CV for all the serum samples showed the high precision of the assay under the same operating conditions. Beyond the repeatability of the SRH assay, this work estimates the effects of random events such as different days and different equipment with the intermediate precision parameter. This parameter was evaluated by performing two different assays by different operators on different analysis sessions in the same laboratory. The CV for each HP, LP and NP human pooled sera was very low and no variation was found in half of the results. The data highlight the great degree of intermediate precision of SRH assay and the capacity to guarantee the same results in different analysis sessions. The precision also includes reproducibility parameter that entails the ability of the assay to provide the same results when performed in different laboratories which has not been evaluated but should be analyzed in further work. The robustness parameter gives an indication of the assay reliability when minor variations occur during testing in a single laboratory. The evaluated variations from the standard procedure were incubation time at different temperatures, initial concentration of the virus, the amount of serum sample and different batches of erythrocytes. We found complete compliance between these variations and the results obtained using standard conditions, highlighting the capacity of the assay to be unaffected by small variations and underlining the high reliability of the assay. Two parameters further estimated are the linearity and accuracy of the assay. The aim of linearity is to illustrate the relationship between concentration and response of the analyte. While the accuracy, evaluated once precision, linearity and specificity have been established, expresses the agreement between the conventional or accepted true value and those found. Our results support the concept of the SRH as a linear and accurate technique in addition to the other features shown above. The stringent demonstration of the SRH assay to meet all regulatory guidelines further increases the advantage of this assay as an easy technique, relatively cheap and allowing the simultaneous testing of a large number of samples without any pre-treatment. In conclusion, this study shows for the first time that the SRH assay is a technique that has the advantage of rapidity and simplicity (Schild et al., 1975) and is not affected by the subjectivity of the operator (Fulton et al., 1984). Furthermore we have confirmed that the SRH is a robust and specific technique for detection of strain specific antibodies against influenza virus. Author contribution All authors contributed to review and revise the manuscript. Conflicts of interest The authors declare no conflict of interest.
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Acknowledgment The work leading to these results has received support from the Innovative Medicines Initiative Joint Undertaking under grant agreement n° 115672, resources of which are composed of financial contribution from the European Union's Seventh Framework Programme (FP7/ 2007-2013) and EFPIA companies’ in kind contribution. References Callow, K.A., Beare, A.S., 1976. Measurement of antibody to influenza virus neuraminidase by single radial hemolysis in agarose gels. Infect. Immun. 13 (1), 1. Chan, Y.C., Tan, H.C., Tan, S.H., Balachandran, K., 1985. The use of the single radial haemolysis technique in the serological diagnosis of dengue and Japanese encephalitis virus infections. Bull. World Health Organ. 63 (6), 1043. Clarke, M., Boustred, J., Seagroatt, V., Schild, G.C., 1977. The use of single-radialhaemolysis for rubella antibody studies. J. Hyg. (Lond.) 79 (3), 355. EMA, 1995. Q2(R1) Validation of Analytical Procedures: Text and Methodology (CPMP/ ICH/381/95). EMA, 1997. Note for Guidance on Harmonisation of Requirements for Influenza Vaccines (CPMP/BWP/214/96). Farrohi, K., Farrohi, F.K., Noble, G.R., Kaye, H.S., Kendal, A.P., 1977. Evaluation of the single radial hemolysis test for measuring hemagglutinin- and neuraminidase-specific antibodies to H3N2 influenza strains and antibodies to influenza B. J. Clin. Microbiol. 5 (3), 353. Food and Drug Administration, 2001. Bioanalytical Method Validation. p. 4. Fulton, R.E., DiNinno, V.L., Frank, R.I., Fildes, J., Turner, I.J., 1984. Single radial hemolysis test for quantitation of complement-fixing antibodies to non-hemagglutinating viruses. J. Clin. Microbiol. 20 (2), 248. Kayali, G., Setterquist, S.F., Capuano, A.W., Myers, K.P., Gill, J.S., Gray, G.C., 2008. Testing human sera for antibodies against avian influenza viruses: horse RBC hemagglutination inhibition vs. microneutralization assays. J. Clin. Virol. 43 (1), 73. McPolin, O., 2009. Validation of Analytical Methods for Pharmaceutical Analysis. Mourne Training Services. Morley, P.S., Hanson, L.K., Bogdan, J.R., Townsend, H.G., Appleton, J.A., Haines, D.M., 1995. The relationship between single radial hemolysis, hemagglutination inhibition, and virus neutralization assays used to detect antibodies specific for equine influenza viruses. Vet. Microbiol. 45 (1), 81. Rowe, T., Abernathy, R.A., Hu-Primmer, J., Thompson, W.W., Lu, X., Lim, W., Fukuda, K., Cox, N.J., Katz, J.M., 1999. Detection of antibody to avian influenza A (H5N1) virus in human serum by using a combination of serologic assays. J. Clin. Microbiol. 37 (4), 937. Russell, S.M., Benjamin, S.R., Briggs, M., Jenkins, M., Mortimer, P.P., Payne, S.B., 1978. Evaluation of the single radial haemolysis (SRH) technique for rubella antibody measurement. J. Clin. Pathol. 31 (6), 521. Schild, G.C., Pereira, M.S., Chakraverty, P., 1975. Single-radial-hemolysis: a new method for the assay of antibody to influenza haemagglutinin. Applications for diagnosis and seroepidemiologic surveillance of influenza. Bull. World Health Organ. 52 (1), 43. Trombetta, C.M., Perini, D., Mather, S., Temperon, N., Montomoli, E., 2014. Overview of serological techniques for influenza vaccine evaluation: past, present and future. Vaccines 2 (4), 707. United States Pharmacopeia, U.S.P., 2008. Validation of Compendial Procedures. p. 1. Wood, J.M., Melzack, D., Newman, R.W., Major, D.L., Zambon, M., Nicholson, K.G., Podda, A., 2001. A single radial haemolysis assay for antibodyto H5 haemagglutinin. Int. Congr. Ser. 1219, 761. World Organisation for Animal Health, O.I.E., 2013. Principles and methods of validation of diagnostic assays for infectious diseases. Manual of Diagnostic Tests and Vaccines for Terrestrial Animals vol. 1.
