Collaborative study to establish a World Health Organization International genotype panel for parvovirus B19 DNA nucleic acid amplification technology (NAT)-based assays.

BACKGROUND AND OBJECTIVES: The aim of the collaborative study was to evaluate a panel of plasma samples containing different genotypes of parvovirus B19 (B19V) for use in nucleic acid amplification technology (NAT)-based assays. MATERIALS AND METHODS: The panel of samples [Center for Biologics Evaluation and Research Parvovirus B19 Genotype Panel 1; National Institute for Biological Standards and Control (NIBSC) code number 09/110] comprises four different members, i.e. Member 1, Member 2, Member 3, and Member 4 (M1-M4); these represent genotypes 1, 2, 3a B19V, and a negative plasma control, respectively. Thirty-five laboratories from 13 different countries participated in the study. Participants assayed the panel members concurrently with the 2nd World Health Organization (WHO) International Standard for B19V DNA (NIBSC code 99/802) on four separate occasions. RESULTS: A total of 44 sets of data were returned, 34 from quantitative assays and 10 from qualitative assays. The majority of assays used were in-house and based on real-time PCR. The results showed that all three genotypes were detected consistently by the majority of participants, although a small number of assays detected genotypes 2 and 3 less efficiently, or not at all. Real-time stability studies have indicated that the panel of B19V samples is stable under normal conditions of storage, i.e. ≤-70°C. CONCLUSIONS: The four-member panel is intended for use in evaluating the ability of NAT assays to detect different B19V genotypes (M1-M3). Based on the results of the collaborative study, the panel was established as the 1st WHO International Reference Panel for parvovirus B19 genotypes.

World Health Organization collaborative study to calibrate the 3rd International Standard for Hepatitis C virus RNA nucleic acid amplification technology (NAT)-based assays.

BACKGROUND AND OBJECTIVES: A collaborative study was undertaken to evaluate a replacement World Health Organization International Standard for hepatitis C virus (HCV) RNA for nucleic acid amplification technology (NAT)-based assays. The candidate preparations were calibrated in International Units (IUs). MATERIALS AND METHODS: Three new candidate preparations were produced from a single bulk containing anti-HCV-negative, genotype 1a HCV RNA-positive plasma. Two samples were lyophilized (coded Sample 2 and Sample 3), whilst a third (Sample 4) contained liquid/frozen material. The samples were distributed together with the 2(nd) International Standard (Sample 1, NIBSC code 96/798) for evaluation by thirty-three laboratories, from fourteen countries. The panel of samples were assayed on four separate occasions. Stability studies were performed for the lyophilized samples by accelerated thermal degradation. RESULTS: Participants returned data from a wide range of commercial and in-house quantitative and qualitative assays. Twenty-five data sets were returned for quantitative assays and fourteen for qualitative assays. Excellent agreement was observed between laboratories and assay methods. The mean relative potencies of Samples 2-4 were 5·19, 5·41 and 5·70 log(10) IU/ml, respectively, when compared against the 2(nd) International Standard. Samples 2 and 3 demonstrated stability of a similar order to the previous standards. CONCLUSIONS: Based upon the results of the collaborative study, Sample 2 (code number 06/100) was established as the 3rd International Standard for HCV RNA with an assigned unitage of 5·19 log(10) IU/ml. Each vial contains the equivalent of 0·5 ml of material; each vial contains 4·89 log(10) IU of HCV RNA.

International collaborative study to establish reference preparations to standardise haemagglutination testing for anti-A and anti-B in normal intravenous immunoglobulins by the direct method.

A joint project (coded BSP089) was run by the European Directorate for the Quality of Medicines & HealthCare (EDQM) of the Council of Europe, the National Institute for Biological Standards and Control (NIBSC) on behalf of the World Health Organization (WHO) and the Center for Biologics Evaluation and Research (CBER) of the U.S. Food and Drug Administration (FDA) to evaluate, in an international collaborative study, 3 lyophilised intravenous immunoglobulin (IVIG) preparations for their suitability to serve as Reference Preparations to standardise and control the highly variable haemagglutination testing for anti-A and anti-B in IVIG products. 23 laboratories tested candidate IVIG reference preparations consisting of a Positive control, a Negative control and a specifically formulated Limit test reference preparation to define the maximum (e.g., pharmacopoeial) limits of anti-A and anti-B haemagglutinins in IVIG products, where limits are applicable. Laboratories performed direct haemagglutination using papain-treated erythrocytes and/or indirect anti-globulin tests. For both methods, there was up to 16-fold variation in anti-A and anti-B titres, although there was good agreement over a 2-fold titre range for anti-A and anti-B between laboratories using the direct method for both the Positive control and Limit reference preparations. Comparative titration data for the Positive control and Limit reference preparations indicated that the use of a 'Limit' test reference preparation would facilitate identification of higher titre batches when the direct haemagglutination method is used. The Positive control, Negative control and Limit test preparations were adopted in November 2008 by the Commission of the European Pharmacopoeia (Ph. Eur.) as Biological Reference Preparations. The same preparations have been established as reference reagents by the WHO and the U.S FDA, including the maximal specifications defined by the Limit test preparation. This will facilitate global standardisation of haemagglutination tests for anti-A and anti-B, ensure that such tests are sufficiently sensitive and specific, and facilitate identification of batches that exceed maximum recommended levels of anti-A and anti-B antibodies.

Collaborative study to establish a replacement World Health Organization International Standard for parvovirus B19 DNA nucleic acid amplification technology (NAT)-based assays.

BACKGROUND AND OBJECTIVES: The aim of the study was to replace the 1(st) World Health Organization International Standard for parvovirus B19 DNA for nucleic acid amplification technique (NAT)-based assays (code 99/800). Two lyophilized preparations (coded 99/800 and 99/802) had been evaluated in the original collaborative study. The present study re-evaluates these two preparations in terms of potency, stability and encapsidation of virus DNA. MATERIALS AND METHODS: The 1(st) International Standard (99/800) and 99/802 were re-coded as Samples 1 and 2, respectively. The samples were distributed to six laboratories and assayed on four separate occasions. Accelerated thermal degradation samples of the two preparations were examined after storage at 20 degrees C for 7 years. Nuclease treatment was used to investigate the encapsidation of virus DNA. RESULTS: Data were returned from a total of six different quantitative NAT-based assays. The results of the present study confirm those of the original, with no significant differences found in estimated international units (IU)/ml for the 1(st) International Standard (Sample 1 in this study) and the proposed replacement preparation, Sample 2 (99/802). Accelerated thermal degradation studies demonstrate that both samples are very stable, with no loss of potency after storage at 20 degrees C for 7 years. Both lyophilized preparations contained the majority of B19V DNA encapsidated in virions. CONCLUSIONS: On the basis of the data presented in this collaborative study, Sample 2 (code number 99/802) was established as the 2(nd) International Standard for parvovirus B19 DNA for NAT-based assays with a potency of 10(6) IU/ml (500 000 IU/vial).

International collaborative study to evaluate candidate reference reagents to standardize haemagglutination testing for anti-A and anti-B in normal intravenous immunoglobulin products.

BACKGROUND AND OBJECTIVES: The aim of the study was to evaluate, in an international collaboration, three lyophilized intravenous immunoglobulin (IVIG) preparations for their suitability to standardize and control haemagglutination testing for anti-A and anti-B in IVIG products. MATERIALS AND METHODS: Twenty-three laboratories tested candidate IVIG reference reagents consisting of a Positive control (07/306), a Negative control (07/308), and a specifically formulated Limit preparation (07/310) to define the maximum (e.g. pharmacopoeial) limits of anti-A and anti-B in IVIG products, where limits are applicable. Laboratories performed direct haemagglutination using papain-treated erythrocytes and/or indirect antiglobulin tests. RESULTS: For both methods, there was up to 16-fold variation in anti-A and anti-B titres, although there was good agreement over a two-fold titre range for anti-A and anti-B between laboratories for both 07/306 and 07/310 using the direct method. Comparative titration data for 07/306 and 07/310 indicated that the use of a 'Limit' reference reagent would facilitate identification of higher titre batches when the direct haemagglutination method is used. CONCLUSIONS: The establishment of preparations 07/306, 07/308 and 07/310 as reference reagents by the World Health Organization will facilitate global standardization of haemagglutination tests for anti-A and anti-B, ensure that such tests are sufficiently sensitive and specific, and facilitate identification of batches that exceed maximum recommended levels of anti-A and anti-B. The Commission of the European Pharmacopoeia and the United States Food and Drug Administration have adopted the same reference reagents including the maximal specifications defined by preparation 07/310.

An international collaborative study to establish the 2nd World Health Organization International Standard for hepatitis B virus DNA nucleic acid amplification technology-based assays.

BACKGROUND AND OBJECTIVES: The aim of this study was to replace the 1st World Health Organization International Standard for hepatitis B virus DNA for nucleic acid amplification technique (NAT)-based assays (code 97/746) with a new International Standard. Two lyophilized preparations freeze dried from the same bulk were evaluated in the original collaborative study (coded 97/746 and 97/750, and termed AA and BB, respectively, in the original study). This present study re-evaluates these two preparations in terms of potency and real-time stability. MATERIALS AND METHODS: The 1(st) International Standard (97/746) and the second lyophilized preparation (97/750) were coded Samples 1 and 2, respectively, in the present study. The samples were distributed to six laboratories and assayed on four separate occasions. Accelerated thermal degradation samples of the two preparations were examined after long-term storage at 4 degrees C and 20 degrees C for more than 51 months. RESULTS: Data were returned from a total of nine different NAT-based assays, five in qualitative format and four in quantitative format. The results of this study confirm the results of the original collaborative study, with no significant differences being found in estimated international units (IU)/ml or polymerase chain reaction-detectable units/ml for the 1(st) International Standard (Sample 1 in this study) and the proposed replacement preparation, Sample 2 (97/750). Real-time and accelerated degradation studies indicate that both samples are very stable. Storage of both preparations at 20 degrees C for more than 51 months resulted in no detectable degradation. CONCLUSIONS: On the basis of the data presented in this collaborative study, Sample 2 (code 97/750) was established as the 2nd International Standard for hepatitis B virus DNA for NAT-based assays with a potency of 10(6) IU/ml (500,000 IU/vial).

International standards for minimum potency of anti-A and anti-B blood grouping reagents: evaluation of candidate preparations in an international collaborative study.

BACKGROUND AND OBJECTIVES: The aim of the study was to evaluate lyophilized monoclonal IgM anti-A and anti-B preparations for use as international standards (IS) to specify recommended minimum potencies of anti-A and anti-B blood grouping reagents in tube tests. MATERIALS AND METHODS: The candidate IS for minimum potency of anti-A and anti-B blood grouping reagents, codes 03/188 and 03/164, respectively, were evaluated against a wide range of commercial anti-A and anti-B blood grouping reagents in an international collaborative study involving 16 laboratories in nine countries. Laboratories titrated 03/188 and 03/164 in parallel with as many commercial anti-A and anti-B blood grouping reagents, respectively, as were available to them, in tube tests according to specified haemagglutination methodology. Three of these laboratories and a further laboratory also titrated 03/188 and 03/164 in parallel with currently available reference preparations for anti-A and anti-B. The ratios of the mean endpoint titres of the anti-A and anti-B reagents to those of 03/188 and 03/164, respectively, within each laboratory were calculated. RESULTS: The ratios of the mean titres of the anti-A reagents to the mean titre of 03/188 within a laboratory fell within 0.062 and 4, i.e. the potencies of the anti-A reagents were between a sixteenth to four times as strong as 03/188. The ratios of the mean titres of the anti-B reagents to the mean titre of 03/164 within a laboratory also fell within 0.062 and 4, with one outlier. CONCLUSIONS: By international consensus, a 1 in 8 dilution of the candidate IS for anti-A, 03/188, and a 1 in 4 dilution of the candidate IS for anti-B, 03/164, were considered appropriate to define the recommended minimum potencies of anti-A and anti-B blood grouping reagents, respectively, in tube tests. On the basis of these results, preparations 03/188 and 03/164 were established by the World Health Organization as International Standards for Minimum Potency of Anti-A and Anti-B Blood Grouping Reagents respectively, and by the US Food and Drug Administration Center for Biologics Evaluation and Research as Minimum Potency Reference Reagents.

An International Standard for specifying the minimum potency of anti-D blood-grouping reagents: evaluation of a candidate preparation in an international collaborative study.

BACKGROUND AND OBJECTIVES: The aim of this study was to evaluate a lyophilized monoclonal immunoglobulin M (IgM) anti-D preparation for use as an International Standard to specify a recommended minimum acceptable potency of anti-D blood-grouping reagents. MATERIALS AND METHODS: The candidate International Standard (99/836) for specifying the minimum potency of anti-D blood-grouping reagents was evaluated against a wide range of commercial anti-D blood-grouping reagents in an international collaborative study involving 20 laboratories in 13 countries. Laboratories titrated reconstituted 99/836, in parallel with as many commercial anti-D blood-grouping reagents as were available to them, in tube tests according to specified haemagglutination methodology for low-protein (e.g. monoclonal IgM) and high-protein (e.g. polyclonal) reagents. The ratios of the mean end-point titres of the reagents to that of 99/836 within each laboratory were calculated. RESULTS: The ratios of the mean titres of the low-protein reagents to the mean titre of 99/836 within a laboratory fell between 0.25 and 2 for 43 of the 45 low-protein anti-D reagents tested (i.e. the potencies of the low-protein reagents compared with 99/836 were between a 1:4 dilution of 99/836 to twice as potent as 99/836). The ratios of the mean titres of the high-protein reagents to the mean titre of 99/836 within a laboratory fell within 0.125 and 1 for eight out of the 10 high protein reagents tested. CONCLUSIONS: By international consensus, a 1:3 dilution of reconstituted 99/836 was deemed appropriate to define a recommended minimum acceptable potency of low-protein anti-D blood-grouping reagents. A 1:8 dilution of reconstituted 99/836 was deemed appropriate to define a recommended minimum acceptable potency of high-protein anti-D blood-grouping reagents. On the basis of the results presented here, 99/836 was established by the World Health Organization as the 1st International Standard for specifying the minimum potency of anti-D blood-grouping reagents, in tube tests.

Standardization of factor VIII and von Willebrand factor in plasma: calibration of the WHO 5th International Standard (02/150).

Calibration of the 5th International Standard factor (F)VIII/von Willebrand factor in plasma (02/150) (5th IS) for five parameters [factor VIII: coagulant activity (FVIII:C); FVIII: antigen (FVIII:Ag), von Willebrand factor: antigen (VWF:Ag), von Willebrand factor: ristocetin cofactor (VWF:RCo), von Willebrand factor: collagen binding (VWF:CB)] was achieved through an international collaborative study involving 37 laboratories. Estimates calculated relative to the previous 4th IS and locally prepared normal plasma pools were not significantly different for estimates of FVIII:Ag, VWF:Ag, VWF:RCo and VWF:CB and hence mean values calculated relative to the 4th IS of 0.94, 0.91, 0.78 and 0.94 IU ampoule(-1), respectively, were assigned. However, estimates for FVIII:C relative to the fresh normal pools (mean 0.61 IU ampoule(-1)) were significantly lower than estimates relative to the 4th IS (mean 0.68 IU ampoule(-1)). In consideration of the good stability of FVIII:C in the 4th IS and the variability of estimates relative to the local pools it was agreed to assign the mean value obtained relative to the 4th IS of 0.68 IU ampoule(-1). For all five parameters the interlaboratory variability (geometric coefficient of variation, GCV%) was larger for estimates calculated relative to the normal pools (range 12.6-16.5%) when compared with estimates calculated relative to the 4th IS (range 3.5-8.3%). An accelerated degradation study performed in six laboratories indicated that the five calibrated parameters are extremely stable when ampoules are stored at -20 degrees C. Mean estimates of predicted loss per year at -20 degrees C ranged from 0% for VWF:CB to 0.029% for VWF:RCo. The 5th IS (02/150) was established by the World Health Organization in November 2003.

Variability in factor VIII concentrate measurement: results from SSC field collaborative studies.

Seven 'field' collaborative studies on factor (F)VIII concentrate potency measurements were carried out, using local routine methodology, standards and calculation of results. Data from five of the 12 different concentrates studied are described in detail. These studies revealed that, for the intermediate-purity and the recombinant FVIII concentrates, one-stage potencies were significantly lower than chromogenic potencies, whilst for the two high-purity FVIII concentrates one-stage potencies were significantly greater than chromogenic potencies. On comparing predilution methods for the intermediate-purity concentrate, equivalent potencies were obtained using either buffer or FVIII-deficient plasma as prediluent. For the two high-purity and the recombinant concentrates, potencies obtained using buffer as prediluent were significantly greater and lower, respectively, than potencies obtained using FVIII-deficient plasma as prediluent. Interlaboratory variabilities were compared over all 12 concentrates studied and coefficients of variation (CVs) for one-stage assays were found to be much greater than for chromogenic assays. This was true for all concentrates except for the intermediate-purity concentrate and samples A and B from the first study, where the reverse was true. Furthermore, much better CVs were obtained when using FVIII-deficient plasma than when using buffer as prediluent, for all FVIII concentrates except for the intermediate-purity concentrate where the reverse was true, and sample B where CVs were equivalent. Overall, CVs were far worse than those obtained in controlled collaborative studies. Generally, however, CVs were better with chromogenic assays and predilution in FVIII-deficient plasma, as is recommended by the International Society on Thrombosis and Haemostasis/Scientific and Standardization Committee, particularly for higher purity and recombinant concentrates.

Analysis of an in vivo assay for inactivated polio vaccine.

Approaches to the statistical analysis of data from the rat bioassay for Inactivated Polio Vaccine are discussed and compared, using data from a recent collaborative study The measured response, an antibody titre obtained from a doubling dilution series, did not satisfy the requirements of normality and homogeneity of variance necessary for the standard parallel line model for quantitative data. Laboratory-specific cut-offs can be defined, to reduce the data to "positive" or "negative" responses, allowing valid analysis with the probit method.

A collaborative study to establish the 6th International Standard for factor VIII concentrate.

A study was carried out to replace the 5th WHO International Standard (IS) for factor VIII concentrate, because of depletion of stocks. Two candidate concentrates (X and Y) were assayed as potential replacements against the 5th IS for FVIII concentrate, in a collaborative study involving 33 laboratories. Collaborators were asked to use the ISTH/SSC recommendations, including pre-dilution of concentrates in FVIII deficient plasma in their assays. Several laboratories performed more than one assay method and altogether there were 21 sets of assays with the one-stage method, 6 with the two-stage method and 26 with the chromogenic method. There was good agreement between laboratories using each method for the comparison of concentrates X and Y against the 5th IS, but the overall potencies by one-stage and chromogenic methods each differed by approximately 5% from the overall mean, with the chromogenic potency approximately 10% higher than the one-stage. Inter-laboratory agreement was slightly better for concentrate Y than X, and stability studies indicated that Y was more stable than X. After considering all the information, together with comments from participants and from the FVIII/FIX Subcommittee of the ISTH/SSC, candidate Y (NIBSC code [97/616]), was proposed and accepted in October, 1998, by the Expert Committee on Biological Standardisation of the World Health Organisation to be the 6th International Standard for Factor VIII Concentrate with an assigned potency of 8.5 IU/ampoule.

Validation of in vitro assays for botulinum toxin: a case study.

Ensuring the reliability and precision of assay results requires careful attention to assay design. In this case study we describe validation studies of an in vitro assay for botulinum neurotoxin type A based on its endopeptidase activity towards immobilised synthetic substrate. This assay, in common with many in vitro assays, is sensitive to changes in reagents and assay conditions and is time dependent. In addition, the toxin is not stable in solution. Differences in estimates of potency, resulting from positional factors, which are not significant in individual assays, are shown to be consistent and statistically significant over a longer series of assays. This study emphasizes that assay validation should not be viewed as a single step in assay development but must be considered as a continuing process if assay results are to be reliable and reproducible.

Calibration and use of an in-house anti-measles IgG standard serum.

For the purpose of research a large quantity of anti-measles IgG working reference serum was needed. A pool of sera from five teenagers was prepared and named Alexandre Herculano (AH). In order to calibrate the AH serum, 18 EIA assays were performed testing in parallel AH and the 2nd International Standard 1990, Anti-Measles Antibody, 66/202 (IS) in a range of dilutions (from 1/50 to 1/25,600). A method which compared parallel lines resulting from the graphic representation of the results of laboratory tests was used to estimate the power of AH relative to IS. A computer programme written by one of the authors was used to analyze the data and make potency estimates. Another method of analysis was used, comparing logistic curves relating serum concentrations with optical density by EIA. For that purpose an existing computer programme (WRANL) was used. The potency of AH relative to IS, by either method, was estimated to be 2.4. As IS has 5000 milli international units (mIU) of anti-measles IgG per millilitre (ml), we concluded that AH has 12,000 mIU/ml.