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Nucleic acid amplification techniques (NAT) are routinely used for clinical diagnostics and monitoring hepatitis B virus (HBV) infections, and are implemented on a voluntary basis for blood screening. A collaborative study was performed to evaluate a replacement WHO International Standard for HBV for the standardization of NAT. Two lyophilised HBV candidates were evaluated by 16 laboratories worldwide, alongside the existing HBV International Standard. The overall mean potency estimates for the candidate samples 1 and 2, relative to sample 3 (2nd HBV International Standard), from quantitative assays, were 5.93 and 5.98 log 10 International Units (IU)/mL respectively. The variability in individual laboratory mean estimates for samples 1-3 for quantitative assays was ∼0.3 log 10 IU/mL. The inter-laboratory variability for qualitative assays was higher. Accelerated thermal degradation studies indicate that both lyophilised candidates are stable and suitable for long-term use. Overall, the results suggested that both candidates were suitable as replacement International Standards. Sample 1 (NIBSC code 10/264) was established as the 3rd WHO International Standard for HBV for NAT with an assigned potency of 850,000 IU/mL (∼5.93 log 10 IU/mL), when reconstituted in 0.5 mL of nuclease-free water. It is intended for the calibration (in IU) of secondary reference materials used in HBV NAT., (Copyright © 2017 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Variability in viral load measurements using nucleic acid amplification techniques (NAT) has a significant impact on the management of Epstein-Barr virus (EBV)-associated diseases, and has highlighted a need for standardisation of these measurements. The aim of this collaborative study was to evaluate the suitability of a range of candidate reference materials to harmonise EBV viral load measurements in a wide range of NAT assays. Candidate materials included lyophilised and liquid whole virus preparations of the EBV B95-8 strain, and preparations of Namalwa and Raji cells. Variability between the individual laboratory mean estimates for each candidate was 2.5 log10 copies/mL. The agreement between laboratories was improved when the potency of each candidate was expressed relative to the lyophilised B95-8 preparation. The results of the study indicate the suitability of this candidate as the 1st WHO International Standard for EBV for NAT. It was established in October 2011 by the WHO's Expert Committee on Biological Standardisation with an assigned potency of 5 × 10(6) International Units (IU) (NIBSC code 09/260). It is intended to be used for the calibration of secondary reference materials, used in EBV NAT assays, in IU, thereby improving the comparability of patient viral load measurements., (Copyright © 2016 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Background: The WHO International Standard (IS) for hepatitis B surface antigen (HBsAg) is used to standardize HBsAg assays. Stocks of the 2nd IS for HBsAg are depleted. The proposal to establish its replacement was endorsed by WHO in 2012., Objective: Preparation of a freeze-dried candidate 3rd IS (NIBSC 12/226); evaluation of its suitability in a WHO international collaborative study; calibration of its potency in International Units (IU)., Study Design: The 3rd IS is based on plasma-derived, purified, inactivated HBsAg from Vietnam. Qualitative and quantitative HBsAg assays were used to evaluate 12/226 alongside the 2nd IS and 1st IS. Blinded study samples included a duplicate of 12/226, a negative control and two diluted plasma samples representing hepatitis B virus (HBV) genotypes A and B., Results: Twelve laboratories from 9 countries returned 22 data sets from 15 methods. The overall geometric mean potency of 12/226 is 47.3IU/mL (±13% CV) when compared to the 2nd IS with HBV subgenotype A2. The 3rd IS has HBV subgenotype B4 with a heterogeneous HBsAg subtype population of ayw1 and adw2. Some genotype-dependent effects on the inter-laboratory variability were observed but overall mean potencies were virtually identical irrespective of the IS used for calibration. Stability studies indicate that the candidate is stable for long-term use., Conclusions: 12/226 was established in October 2014 by the WHO Expert Committee on Biological Standardization as the 3rd IS for HBsAg with a potency of 47.3IU per ampoule maintaining the continuity in the standardization of HBsAg assays., (Copyright © 2016 The Authors. Published by Elsevier B.V. All rights reserved.)

Background: Interassay harmonization of cytomegalovirus (CMV) DNA measurement is important for infection management. Uncertainty exists regarding the result harmonization achievable in patient plasma samples using quantitative polymerase chain reaction (qPCR) assays with calibrators now traceable to the First World Health Organization International Standard (IS) for CMV DNA., Method: Serial dilutions of the IS and a blinded panel of 40 genotyped CMV DNA-positive pooled plasma samples and 10 negative plasma samples were tested by 6 laboratories using 10 qPCR assays calibrated to the IS. Each clinical sample was constructed using plasma from a single unique transplant recipient., Results: The variance for individual CMV DNA-positive samples was greater for clinical samples (median, 1.50 [range, 1.22-2.82] log10 IU/mL) than for IS dilutions (median, 0.94 [range, 0.69-1.35] log10 IU/mL) (P < .001); 58.9% of all clinical sample results and 93.6% of IS dilution results fell within ±0.5 log10 IU/mL of the mean viral load of each sample. Result variability was not impacted by either genotype or quantitative levels of CMV DNA. Testing procedure differences can significantly influence results, even when analyte-specific reagents are identical. For clinical samples, all assays demonstrated result bias (P < .008). Assays with amplicon sizes ≤86 bp had significantly higher results compared to assays with larger amplicon sizes (≥105 bp) (P < .001)., Conclusions: The variability in CMV DNA results reported on individual samples has been reduced by the IS, but ongoing clinically relevant variability persists, preventing meaningful interassay result comparison., (© The Author 2016. Published by Oxford University Press for the Infectious Diseases Society of America. All rights reserved. For permissions, e-mail journals.permissions@oup.com.)

Variability in the performance of nucleic acid amplification technology (NAT)-based assays presents a significant problem in the diagnosis and management of human cytomegalovirus (HCMV) infections. Here we describe a collaborative study to evaluate the suitability of candidate reference materials to harmonize HCMV viral load measurements in a wide range of NAT assays. Candidate materials comprised lyophilized Merlin virus, liquid Merlin virus, liquid AD169 virus, and purified HCMV Merlin DNA cloned into a bacterial artificial chromosome. Variability in the laboratory mean HCMV concentrations determined for virus samples across the different assays was 2 log10. Variability for the purified DNA sample was higher (>3 log10). The agreement between laboratories was markedly improved when the potencies of the liquid virus samples were expressed relative to the lyophilized virus candidate. In contrast, the agreement between laboratories for the purified DNA sample was not improved. Results indicated the suitability of the lyophilized Merlin virus preparation as the 1st WHO International Standard for HCMV for NAT. It was established in October 2010, with an assigned potency of 5 × 10(6) International Units (IU) (NIBSC code 09/162). It is intended to be used to calibrate secondary references, used in HCMV NAT assays, in IU., (Copyright © 2016 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Two candidate preparations of human sequence recombinant Interleukin-2 (IL-2) were formulated and lyophilized at NIBSC prior to evaluation in a collaborative study for their suitability to serve as a replacement international standard. The preparations were tested by eight laboratories using in vitro bioassays and immunoassays. The candidate preparation 86/500 was judged suitable to serve as a replacement international standard based on the data obtained for activity and stability. On the basis of the results reported here, the preparation coded 86/500 was established by the WHO Expert Committee on Biological Standardisation (ECBS) in 2012 as the WHO 2nd IS for human IL-2 with an assigned value for IL-2 activity of 210IU/ampoule. Calibration of the 2nd IS is primarily based on the bioassay in use in various laboratories and relies exclusively on the estimates calculated relative to the WHO 1st IS for continuity of the IU., (© 2013. Published by Elsevier B.V. All rights reserved.)

One candidate preparation of human sequence recombinant transforming growth factor-β3 (TGF-β3) was formulated and lyophilized at NIBSC prior to evaluation in a collaborative study for its suitability to serve as an international standard. The preparation was tested by 8 laboratories using in vitro bioassays and immunoassays. The candidate preparation 09/234 was judged suitable to serve as an international standard based on the data obtained for biological activity and stability. On the basis of the results reported here, the preparation coded 09/234 was established by the WHO Expert Committee on Biological Standardisation (ECBS) as the WHO 1st IS for human TGF-β3 with an assigned value for TGF-β3 activity of 19,000 IU/ampoule., (Copyright © 2012 Elsevier B.V. All rights reserved.)

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., (© 2011 The Author(s). Vox Sanguinis © 2011 International Society of Blood Transfusion.)

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., (© 2010 The Author(s) Vox Sanguinis © 2010 International Society of Blood Transfusion.)

Five candidate preparations of human sequence recombinant granulocyte-colony stimulating factor (G-CSF) were formulated and lyophilized at NIBSC prior to evaluation in a collaborative study for their suitability to serve as a replacement International Standard (IS). The preparations were tested by 13 laboratories using in vitro bioassays. The candidate preparation 09/136 was judged suitable to serve as a replacement international standard based on the data obtained for biological activity and stability. On the basis of the results reported here, the preparation coded 09/136 was established by the WHO Expert Committee on Biological Standardization (ECBS) as the 2nd IS for human G-CSF with an assigned value for G-CSF activity of 95,000 IU/ampoule. Calibration of the 2nd IS is primarily based on the bioassay in use in various laboratories and relies entirely on the estimates calculated relative to the WHO 1st IS for continuity of the IU., (Copyright © 2011 Elsevier B.V. All rights reserved.)

A World Health Organization collaborative study was conducted to evaluate candidate international standards for human papillomavirus (HPV) Type 16 DNA (NIBSC code 06/202) and HPV Type 18 DNA (NIBSC code 06/206) for use in the amplification and detection steps of nucleic acid-based assays. The freeze-dried candidate international standards were prepared from bulk preparations of cloned plasmid containing full-length HPV-16 or HPV-18 genomic DNA. Nineteen laboratories from 13 countries participated in the study using a variety of commercial and in-house quantitative and qualitative assays. The data presented here indicate that, upon freeze-drying, there is no significant loss in potency for the candidate HPV-18 DNA and a slight loss in potency for the candidate HPV-16 DNA; although this is likely not scientifically relevant when assay precision is considered. In general, the individual laboratory mean estimates for each study sample were grouped +/- approximately 2 log(10) around the theoretical HPV DNA concentration of the reconstituted ampoule (1 x 10(7) HPV genome equivalents/mL). The agreement between laboratories is improved when potencies are made relative to the candidate international standards, demonstrating their utility in harmonizing amplification and detection steps of HPV-16 and -18 DNA assays. Degradation studies indicate that the candidate international standards are extremely stable and suitable for long-term use. Based on these findings, the candidate standards were established as the 1st WHO international standards for HPV-16 DNA and HPV-18 DNA, each with a potency of 5 x 10(6) international units (IU) per ampoule or 1 x 10(7) IU mL(-1) when reconstituted as directed.

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.

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).

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.

Background: In order to harmonize results for the detection and quantification of Plasmodium falciparum DNA by nucleic acid amplification technique (NAT)-based assays, a World Health Organization (WHO) collaborative study was performed, evaluating a series of candidate standard preparations., Methods: Fourteen laboratories from 10 different countries participated in the collaborative study. Four candidate preparations based upon blood samples parasitaemic for P. falciparum were evaluated in the study. Sample AA was lyophilized, whilst samples BB, CC and DD were liquid/frozen preparations. The candidate standards were tested by each laboratory at a range of dilutions in four independent assays, using both qualitative and quantitative NAT-based assays. The results were collated and analysed statistically., Results: Twenty sets of data were returned from the participating laboratories and used to determine the mean P. falciparum DNA content for each sample. The mean log10 "equivalents"/ml were 8.51 for sample AA, 8.45 for sample BB, 8.35 for sample CC, and 5.51 for sample DD. The freeze-dried preparation AA, was examined by accelerated thermal degradation studies and found to be highly stable., Conclusion: On the basis of the collaborative study, the freeze-dried material, AA (NIBSC code No. 04/176) was established as the 1st WHO International Standard for P. falciparum DNA NAT-based assays and has been assigned a potency of 10(9) International Units (IU) per ml. Each vial contains 5 x 10(8) IU, equivalent to 0.5 ml of material after reconstitution.

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).

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.

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.

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.

Folate measurements, particularly for whole blood, show wide inter-laboratory and inter-methodology variability. This variability appears to be due in part to the lack of internationally accepted reference materials. A whole blood haemolysate, lyophilised in ampoules and designated 95/528, was therefore evaluated by 15 laboratories in five countries for its suitability as an International Standard (IS) for whole blood folate. The preparation was assayed using a variety of microbiological and protein-binding methodologies against local standards and calibrators. A consensus folate content was assigned to 95/528. The inclusion of three whole blood samples in the study with widely differing folate levels demonstrated a considerable reduction in inter-laboratory variability when the folate content of the samples was determined relative to the proposed IS 95/528 rather than to laboratories' local standards and calibrators. Accelerated degradation studies indicated that the folate content of 95/528 is stable when stored at -20 degrees C. On the basis of the results presented here, the World Health Organization Expert Committee on Biological Standardization established 95/528 as an IS for whole blood folate.

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.

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 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.

Eighteen laboratories participated in a collaborative study to calibrate a replacement for the 1st International Standard for Antithrombin. Concentrate (88/548). Excellent agreement between laboratories, as indicated by mean % gcv of 3.3 and 5.9 for functional and antigenic assays, was observed when the candidate concentrate (96/520) was assayed against the 1st International Standard for Antithrombin, Concentrate (88/548). The functional potency was found to be 7.9% (p<0.05) lower than the antigenic potency. Based on the results and with the agreement of the participants of this study and the authorisation of the Expert Committee on Biological Standardisation of the World Health Organisation, the antithrombin concentrate, coded 96/520, has been established as the 2nd International Standard for Antithrombin, Concentrate, with labelled potencies for both functional (4.7 IU/ampoule) and antigenic (5.1 IU/ampoule) activities.

One hundred randomly chosen sera from blood donors from North London were assayed for antibodies to mumps virus by plaque reduction and microtitre neutralisation assay, haemagglutination inhibition and in-house ELISA. The assay reproducibility was determined, and there was reasonable agreement between antibody levels measured by the two neutralising methods. Neutralising antibody levels measured by either method were low but the strain used in the assay had a large effect on the antibody titres observed. Titres measured by neutralisation assay, HI assay and ELISA did not correlate well. Assessment of immunity to mumps virus remains problematical.

An international collaborative study to assess inter-laboratory variation in the sensitivity of gene amplification assays for the detection of HIV-1 RNA sequences was conducted using a panel of eight duplicate dilutions of an HIV-1 genotype B clinical isolate and negative control samples. Twenty-five laboratories participated in the study and used a variety of in-house assays and commercial assay systems. With few exceptions, the assays were more sensitive than a p24 antigen assay. Overall, the PCR-based Amplicor Monitor assay was the most sensitive and gave the highest mean copy number for any one sample. Some of the in-house assays gave results comparable with the Monitor assay whilst the NASBA and bDNA assays appeared to be less sensitive. As a result of this study, an HIV-1 Working Reagent for the standardisation of nucleic acid amplification assays was developed and assessed in a subsequent study. Similar differences in sensitivity between the different assay systems was observed. The discrepancies in viral copy number obtained using the Working Reagent highlights the need for an International Standard against which all Working Reagents may be calibrated.

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.

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.

Assays of the potency of inactivated poliovirus vaccine require the use of an appropriate reference reagent. Preparation 91/574 was shown by international collaborative study to be suitable for determination of antigenic content and immunogenicity of inactivated poliovirus vaccines by in vitro and in vivo assays, respectively. The reagent is a trivalent blend of formaldehyde-inactivated monovalent pools of poliovirus type 1 (Mahoney) poliovirus type 2 (MEF-1) and poliovirus type 3 (Saukett). Studies by antigen-capture ELISA showed that the component monovalent pools contained high titres of D antigen and trace amounts of C antigen. Sucrose gradient analysis showed that the D antigenicity was almost exclusively associated with 150S virus particles. Low levels of procapsids (75S particles with D antigenicity) were detected in the type 1 and 2 monovalent pools. The profile of intact virions and procapsids in 91/574 in sucrose gradients was very similar to PU78-02, a previously used inactivated trivalent poliovirus vaccine reference. The World Health Organization (WHO) Expert Committee on Biological Standardization at its 1994 meeting established preparation 91/574 as the 2nd WHO international Reference Reagent for poliomyelitis vaccine (inactivated). A potency of 430, 95, 285 D antigen units per ml was assigned to poliovirus type 1, 2 and 3, respectively. A separate aliquot of this preparation, established by the European Pharmacopeia Commission as a Biological Reference Preparation, has an identical assigned titre. The 2nd WHO International Reference Reagent 91/574 is intended for calibration of secondary reference reagents.

The nucleotide sequences of the SH gene of 45 new mumps virus isolates derived from different parts of Europe, Canada and USA were determined. A phylogenetic tree was constructed which confirmed the existence of three major clusters. While clustering according to geographical origin was clear in some cases each of the major clusters included isolates which were widely separated in origin. The degree of variability between the sequences of SH genes of different strains and the ratio of coding to non coding differences were both very high compared to those observed in other genes such as the M and HN. A standardized system of nomenclature of mumps strains was established.

Ten laboratories studied the immunogenicity of six trivalent inactivated poliovirus vaccines in a WHO Collaborative Study. The antigenic content of all six vaccines had previously been estimated in an earlier study (Wood et al., 1995). In collaboration with the European Pharmacopoeia Commission an additional preparation, European Pharmacopoeia Biological Reference Preparation Batch 1, was also included. Laboratories were requested to use established immunogenicity assays and six used the European Pharmocoepia guinea pig/chick test while three used a rat potency test. One laboratory contributed data from both methods. Apart from one laboratory, within laboratory variation was low (less than five-fold). However, very large (greater than 100-fold) variation was seen between laboratories for ED50 results in the guinea pig/chick test. Different decisions on pass/fail outcome would have occurred for some of the samples tested. Between laboratory variation was much lower (less than five-fold) in the rat test. Expression of results as potencies relative to a standard reduced between laboratory variation for both methods, substantially so for the guinea pig/chick test. The correlation between in vivo and in vitro results was generally good with the exception of the type 3 component of one preparation. This showed that the relationship between immunogenicity and antigenicity was not necessarily predictable. There is an urgent need to revise the European Pharmocoepia immunogenicity test but it is premature, on the basis of this study, to recommend either the rat test or in vitro tests as replacements. Two candidate reference materials were both found suitable for in vivo assay of inactivated poliovirus vaccine.

A European collaborative study, in which 16 laboratories participated, was carried out to assess the performance of the European Pharmacopoeia (EP) monograph methods for anticoagulant activities (anti-Xa and anti-IIa assays) of low molecular mass (LMM) heparin and to assess the suitability of six candidate materials as the EP working standard for LMM heparin. There was good interlaboratory agreement for both types of assays as indicated by most gcy's being less than 10%, indicating acceptable performance of the EP assay methods. All the candidate preparations gave dose-response curves parallel to the 1st International Standard for Low Molecular Weight heparin and to each other. All preparations, possibly with the exception of E and F, gave similar performance as measured by interlaboratory agreement and would be suitable as working standards. Based on these data, preparations A, B, C and D have been established by the EP as official EP Biological Reference Preparations and they will be issued as successive batches.

In 1988, a collaborative study was set up to examine the sensitivity and reproducibility of assays for the detection of DNA in biologicals derived from continuous cell lines. Fifteen laboratories analyzed 12 samples containing different amounts of DNA and protein. When no or very little DNA was present in the test samples, false positives or overestimation was common. In contrast, when higher levels of DNA were present underestimation was the norm. The study also revealed a high degree of variability between laboratories.

A competitive radioimmunoassay using monoclonal anti-D antibodies has been developed for measuring anti-D activity in prophylactic immunoglobulin preparations. The assay is totally specific for anti-D, reproducible and gives potency estimates comparable to those determined using autoanalyzer methodology. The assay also provides a means for comparing different batches of monoclonal anti-D preparations for red-cell binding activity.

In the first phase of a two part WHO Collaborative study, fourteen laboratories from ten countries estimated the antigenic content of six trivalent inactivated poliovirus vaccine preparations using in vitro methods. All laboratories used a candidate standard method for D antigen assay (method A) and eight contributed results from established 'in-house' methods (method B). All methods assayed D antigen in an antigen capture ELISA format. Monoclonal antibodies were used as detector reagents in method A and in some laboratories for method B. The average difference in potency estimates for duplicate preparations A and C was used to assess within assay variation. Overall this was found to be 22% and 19% for methods A and B respectively. Within laboratory variation was measured as the geometric coefficient of variation for between assay repeatability. Results for methods A and B, 28% and 26% respectively were again very similar. Variation in potency estimates between laboratories was in the range 2- to 5-fold for most samples and most laboratories irrespective of the method used. However, a maximum 24-fold difference occurred when all results were taken into account. Method A gave significantly enhanced potency estimates for the type 3 component of preparation B, a vaccine shown to be immunogenic in humans in clinical trials, compared to method B. Method A also failed to assay the type 3 component of preparation F which was prepared by inactivation of the Sabin 3 strain of poliovirus. Further work is required to identify monoclonal antibodies, or combinations of monoclonal antibodies, suitable for universal application in D antigen assays of inactivated poliovirus vaccines. Further work is also required to improve control of the antigen-capture ELISA in some laboratories. The second phase of this WHO Collaborative Study evaluated the proficiency of in vivo potency assays. These results together with an evaluation of the correlation of immunogenicity and antigenic assay plus assessment of candidate reference materials will be reported separately.

A collaborative study evaluated the histopathological examination of the WHO neurovirulence test for poliovirus vaccines. Participants scored two sets of slides and the slides were sent to all participants in turn. The results showed an approximately two-fold range in mean lesion scores between readers. As readers scored consistently high or low, however, the range of differences between the two sets of slides was considerably less. From the limited data available, these differences in scoring would not have resulted in different decisions on the acceptability of the particular vaccines included in this study. The study was of value in assisting in developing mutual confidence in the interpretation of histological lesions in the neurovirulence test.

A collaborative study involving 10 European laboratories was undertaken to assess the variability of estimates of the potency of measles, mumps and rubella tri-valent vaccines. The precision of assays as demonstrated by tests on duplicate samples was good; differences averaged around 0.2 log10 steps. Similarly, assay to assay variation within laboratories was small with most achieving consistency around 5% over three assays. In contrast, overall median variations in potency between laboratories were around 1.0 log10 for measles, 3.0 log10 for mumps and 2.0 log10 for rubella. Unexpectedly, the variations in estimates for measles and rubella were not improved when potencies were expressed relative to reference preparations. However, for mumps variability was reduced by using a reference but only for the vaccines of the same strain as the reference. For these Urabe mumps vaccines the variation in relative potency was around 1.5 log10.