3rd International Standard Research Articles

Analytical Performance of the Sysmex HISCL HBsAg Assay and Comparison with the Roche Elecsys HBsAg II Quant Assay in the Quantification of Hepatitis B Surface Antigen.

Background and Objectives: This study aims to estimate the analytical performance of the Sysmex HISCL HBsAg assay and to assess the analytical correlation with the Roche Elecsys HBsAg II quant assay with clinical samples and the WHO International Standard (IS). Materials and Methods: The intra-assay precision, linearity, assay limitation, accuracy, and comparative evaluation of the HISCL HBsAg assay were estimated. Results: Extrapolating from the plot of the average total allowable error versus the reference value, an accuracy goal of 20% would be achieved around a limit of quantification (LoQ) of 0.014867 IU/mL. The percentage of biases for each level of the WHO IS measured by the two assays were less than 15%, except for the WHO 3rd IS, for which the HISCL HBsAg assay achieved a percentage of bias of 33%. In the comparative evaluation, Passing–Bablok regression analysis did not reveal any significant deviation from linearity between the two assays (y = −48.6998 + 1.9206x; p = 0.79 by the CUSUM test for linearity). The mean difference of the quantitative HBsAg level between the two assays was 1762.5 IU/mL in the Bland–Altman plot. Conclusions: The HISCL HBsAg assay, with a highly sensitive LoQ of 0.03 IU/mL, showed similar analytical performance in HBsAg quantification to the Elecsys HBsAg II quant assay and may be helpful in obtaining better diagnoses and therapeutic strategies for treating HBV infections.

Do Australian immunoglobulin products meet international measles antibody titer standards?

ABSTRACTThe effectiveness of passive immunisation post-exposure to measles appears subject to a dose-response effect. New Zealand and the United Kingdom have increased the recommended dose of polyclonal human immunoglobulin for post-exposure prophylaxis within the last decade in response to concerns about decreasing levels of measles antibodies in these products. This study used the plaque-reduction neutralization test (PRNT) to measure the titer of measles-specific antibodies in Australian immunoglobulin products for post-exposure prophylaxis and compared the utility of an enzyme-linked immunosorbent assay (ELISA) to the PRNT in available Australian and international samples: Australian intramuscular (n = 10), Australian intravenous (n = 28), New Zealand intramuscular (n = 2), Hizentra (subcutaneous)(USA) (n = 3), and Privigen (intravenous)(USA) (n = 2). Measles titres in Australian IM and IV immunoglobulins ranged from 51 to 76 IU/mL and 6 to 24 IU/mL respectively, as measured by PRNT calibrated to the WHO 3rd international standard. ELISA titres were variable but higher than PRNT titres in all tested samples. Measles antibody titres in Australian immunoglobulin products meet consensus-prescribed international thresholds. Development of a convenient, standardized, readily accessible assay for determination of measles titres in immunoglobulin products would be useful for future studies and facilitate international comparisons.

Collaborative study for the calibration of a replacement International Standard for diphtheria toxoid for use in flocculation test

We present the results of a collaborative study for the establishment of a replacement International Standard (IS) for diphtheria toxoid for use in flocculation test and its calibration in Lf units. Calibration was performed using Ramon flocculation method, standardized using the 2nd IS. The candidate standard was assigned a unitage of 1870 Lf/ampoule based on results from 25 laboratories in 15 different countries and was established as the 3rd IS for diphtheria toxoid for use in flocculation test by the WHO Expert Committee on Biological Standardization (ECBS) in October 2015.The study also assessed the use of alternative methods for measuring Lf. Participants were asked to determine the Lf value of the candidate standard using an Enzyme Linked Immunosorbent Assay (ELISA) established at NIBSC, or other suitable in-house method. 10 laboratories performed ELISA according to the NIBSC protocol, 1 laboratory performed flocculation using laser-light scattering according to an in-house protocol, and 1 laboratory performed another in-house ELISA. Results suggest these methods may provide suitable alternatives to the Ramon flocculation test, subject to validation, and that the new standard could act as a suitable reference preparation in these methods.

Establishment of replacement International Standard 13/132 for human antibodies to Toxoplasma gondii

Sixteen laboratories carried out a collaborative study to validate 13/132 as a replacement International Standard (IS) for TOXM (3rd IS for anti-Toxoplasma Serum, Human, 1000 IU). 13/132 is a freeze dried preparation of pooled human plasma from six donors who experienced a recent Toxoplasma gondii infection. The potency of 13/132 was compared to TOXM and 01/600 (1st IS for anti-Toxoplasma IgG, Human, 20 IU). Samples were tested for IgA, IgG, IgG avidity and IgM in agglutination assays; enzyme linked immunosorbent assays (ELISA), enzyme linked fluorescent assays, immunoblots, immunofluorescence assays and the Sabin–Feldman dye test for Ig. 13/132 was strongly positive for Ig, IgA, IgG and IgM and the reproducibility was very good. 13/132 contains high levels of anti-Toxoplasma Ig, IgG and IgM and its potency falls between TOXM and 01/600. The avidity of IgG was found to be low, similar to the avidity of IgG from TOXM. 13/132 was established by the Expert Committee on Biological Standardization as the 4th IS for Antibodies, Human, to T. gondii with an assigned unitage of 160 IU per ampoule for Ig by dye test and 263 U per ampoule for IgG by ELISA.

International collaborative study on the 3rd WHO International Standard for hepatitis B surface antigen

BackgroundThe 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. ObjectivePreparation 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 designThe 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. ResultsTwelve 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. Conclusions12/226 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.

Characterization of the 3rd International Standard for hepatitis B virus surface antigen (HBsAg)

BackgroundHBsAg is the most important marker for laboratory diagnosis of HBV infection. Validation and quality control of HBsAg tests requires International Standards (IS). Recently the 2nd IS was replaced by the 3rd IS. Both IS are made from plasma-derived hepatitis B vaccines, but production and geographical origin are different. ObjectiveCharacterization of the HBsAg in the source material (SM) for the 3rd IS and comparison with the 2nd IS and native HBsAg. Study designThe SM was analyzed using solid-phase immunoassays, quantitative immune electrophoresis, ultracentrifugation, immunoblotting and HBV DNA sequencing. ResultsThe plasma-derived HBsAg of the SM originated from at least two different HBV strains, both of subgenotype (sgt) B4, typical for Vietnam. The HBsAg subtype was heterogeneous with ayw1 and adw2. The HBsAg concentration was 23,700 IU/ml as determined by solid-phase immunoassay; immune electrophoresis calibrated with sgt B2 revealed a concentration of 24,500 IU/ml while calibration with sgt D1 provided lower values. Proteins in the SM are heterogeneous in size containing only traces of preS. The protein subunits are partially cross-linked. ConclusionsThe antigenicity of the 3rd IS is suitable for HBsAg calibration in laboratory tests. In contrast to the 2nd IS, the 3rd IS is representative for a highly endemic region. Similar to the 2nd IS and different from native HBsAg, preS domains are depleted, protein subunits are partially cross-linked and the HBsAg particles are partially aggregated in the 3rd IS. The HBV subgenotype differences between the two IS may lead to variations in different quantitative assays.

An international collaborative study to investigate a proposed reference method for the determination of potency measurements of fibrinolytics in absolute units.

Traditionally, WHO International Standards (IS) have been calibrated in International Units (IU) by consensus following an international collaborative study. In the area of coagulation and fibrinolysis standards it is also common for laboratories involved in such studies to perform their own in-house methods, although guidelines may be defined to include recommendations of replication and randomization of sample testing to improve the robustness of the study. The historic basis of this approach has been to develop a common reference standard to facilitate comparisons of results for the relative potency of standard and test preparations in laboratories using different methods [1]. However, this approach has been criticized and suggestions for improvements have been made which would bring the standardization of biologicals more in line with other calibrators used in medicinal chemistry. Guidelines for the introduction of a metrologically sound approach to standardization have been detailed elsewhere [2]. General goals include standardization of methods and the introduction of a hierarchy of reference materials and procedures, each with an assigned uncertainty, to provide a system of metrological traceability where testing of routine samples can ultimately be traced back to a primary calibrator and primary reference method that are defined in SI units [3]. We have previously published a proposal for a reference method developed to measure the potency of thrombolytic products (plasminogen activators) [4] that did allow the calculation of activity in enzyme units (moles of product per second in the defined method), which could theoretically be converted into katals. The katal (mole per second) is the coherent derived SI unit of measurement for enzyme activity and is at the top of the hierarchy for the catalytic concentration of an enzyme [5]. Thus it would be possible theoretically to define the concentration of an IS not only in IU but also by absolute SI units. With the encouragement of the Fibrinolysis SSC, an international collaborative study was organized in which laboratories expert in fibrinolysis methods were recruited to perform the defined method [4] using the current IS for urokinase (uPA, 87/594), tissue plasminogen activator, (tPA, 98/714) and streptokinase (SK, 00/464). The study was planned as far as possible to remove possible sources of variation. All necessary reagents were provided, including IS, plasminogen substrate (NIBSC reagent 97/534), and chromogenic substrate for plasmin CS-41(03) (Hyphen BioMed, France). In addition, thrombin (NIBSC reagent 01/578) and fibrinogen concentrate (1st IS 98/614) were provided in order to make clots as consistently as possible in all laboratories. Plasmin (3rd IS, 97/ 536) was also provided to all laboratories to perform a series of assays in which a range of concentrations of chromogenic substrate was hydrolysed completely in order to calculate an extinction value for p-nitroaniline for each laboratory that was specific to the equipment they used. This value was critical to calculate the molar concentration of p-nitroaniline released during the plasminogen activation reaction, which in turn allows the molar concentration and rate of plasmin generation to be calculated and thus express the activity of the plasminogen activator in SI units. The only materials provided by the laboratories in the study were Tris buffer and microtitre plates. A detailed collaborative study protocol was agreed in conjunction with participating laboratories and other outside interested parties over a series of months ahead of the practical phase of the study. Twelve participants contributed a total of 36 assays, each of which included the three activators, uPA, tPA and SK at four doses, in quadruplicate for each point. Raw data of absorbance vs. time were returned to NIBSC for analysis, where rates of plasminogen Correspondence: C. Longstaff, Haemostasis Section, National Institute for Biological Standards and Control, South Mimms, Herts, EN6 3QG, UK. Tel.: +44 1707 641253; fax: +44 1707 641050; e-mail: clongstaff@ nibsc.ac.uk

Biological standardization of human interferon beta: Establishment of a replacement world health organization international biological standard for human glycosylated interferon beta

Human interferon beta (IFN-β) has been developed as a major biotherapeutic agent for the treatment of multiple sclerosis. Since World Health Organization (WHO) international standards (IS) for IFN-β were established several years prior to the development of clinical grade IFN-β products, a number of scientific issues with regard to the biological standardisation of natural and recombinant IFN-β products have emerged. In order to address these issues, an international collaborative study to evaluate WHO IS and candidate international standards (CIS) of IFN-β was instigated by the National Institute for Biological Standards and Control (NIBSC) in 2000 and was carried out in the succeeding year. Sixteen expert laboratories from 8 countries worldwide participated in the study. They performed titrations on 8 different IFN-β preparations, including IS and new CIS, in a variety of mainly antiviral- but also including some antiproliferative- and reporter gene-assays, and contributed raw data from these assays to NIBSC for statistical analysis and calculation of potencies. While both intra- and inter-laboratory variation of potency estimates was evident, overall validity of the study as a whole was clearly shown by comparison of two pairs of internal coded duplicates, which gave the expected relative potency of 1 and the lowest inter-laboratory variability of potency estimates in all assay types. The CIS containing Chinese hamster ovary (CHO) cell- or human fibroblast-derived, glycosylated, IFN-β gave similar low inter-laboratory variation in potency estimates one to another as the coded duplicates, which was significantly less than to the 2nd WHO IS of IFN-β, human fibroblast-derived, Gb23-902-531. One of these CIS, designated 00/572, containing CHO cell-derived IFN-β and formulated with both bovine casein and human serum albumin, could be assigned a potency, consistent for all assay types, of 40,000 international units (IU) per ampoule relative to the IU of the 2nd IS of IFN-β, Gb23-902-531. Other CIS containing glycosylated IFN-β, either CHO cell- or human-fibroblast-derived, could also be assigned potency values that were continuous with the IU of Gb23-902-531 and 00/572. However, greater inter-laboratory variations in estimates were evident from comparisons of Gb23-902-531 or 00/572 with either the 1st IS for E. coli-derived, non-glycosylated, IFN-β with serine substitution at position 17 (IFN-β Ser 17 mutein), Gxb02-901-535, or with a CIS (00/574) containing IFN-β Ser 17 mutein. Indeed, variations in potency estimates for preparations containing IFN-β Ser 17 mutein were sufficiently large to indicate that assays could distinguish preparations of IFN-β Ser 17 mutein from preparations of glycosylated IFN-β. Thus, neither the 2nd IS of IFN-β, Gb23-902-531, containing fibroblast-derived IFN-β, nor CIS, 00/572, containing CHO cell-derived IFN-β, was appropriate for standardisation of preparations of IFN-β Ser 17 mutein. Conversely, neither the IS of IFN-β Ser 17 mutein, Gxb02-901-535, or a CIS of IFN-β Ser 17 mutein, 00/574, was appropriate for the standardisation of preparations of glycosylated IFN-β. CIS 00/572, containing CHO cell-derived, glycosylated IFN-β, was clearly shown to be suitable to serve as a primary standard for glycosylated forms of IFN-β, especially clinical grade IFN-β-1a products. It was further shown to exhibit high thermal and long-term stability. Since the CHO cell-derived IFN-β used for preparation of 00/572 was of a greater purity than the IFN-β used for the 2nd IS of IFN-β, Gb23-902-531, it was recommended by the WHO Informal Consultation on the Standardisation of Cytokines, Growth Factors and Other Endocrinological Substances, which met in October 2003, that 00/572 should replace Gb23-902-531 as the IS for glycosylated IFN-β. This recommendation was accepted by the WHO Expert Committee on Biological Standardization (ECBS) at its annual meeting in November 2003 and 00/572 was established as the 3rd IS for human glycosylated IFN-β with an assigned potency of 40,000 IU. As this study identified no advantage to replacing the existing 1st IS for IFN-β Ser 17 mutein, Gxb02-901-535, WHO ECBS accepted that this should continue to serve as the IS for this material.

International collaborative study to establish the 3rd International Standard for Streptokinase

An international collaborative study was organized to calibrate a replacement for the current (2nd) International Standard (IS) for Streptokinase, stocks of which are almost exhausted. Two candidate preparations were assayed against the 2nd IS in a study involving 16 laboratories in 12 countries: preparation 88/824 (coded B), and preparation 00/464 (C and D, coded duplicates). Laboratories could use two methods provided, either a fibrin clot lysis assay or a solution chromogenic method, or an in-house method. Laboratories were encouraged to perform more than one method if possible. With the exception of one laboratory which gave outlying results for preparation 00/464, there was good agreement within and between laboratories and no significant differences between potencies using the different methods employed. This study demonstrates that a solution chromogenic assay is an acceptable format for potency determination of the streptokinase preparations in this study and fibrin is not necessary. It has now been agreed that a solution chromogenic plasminogen activation assay replace the current euglobulin reference method for streptokinase activity determination in the European Pharmacopoeia. Study participants, SSC of the International Society on Thrombosis and Haemostasis and the Expert Committee on Biological Standardization (ECBS) at the World Health Organization approved preparation 00/464 (C,D in the study) as the 3rd IS for Streptokinase with a potency of 1030 IU per ampoule.

Standardisation of Factor VIII and von Willebrand Factor in Plasma: Calibration of the 4th International Standard (97/586)

The 4th International Standard (IS) Factor VIII/von Willebrand Factor (FVIII/VWF) plasma was calibrated in 25 laboratories by assay against the 3rd IS plasma and fresh normal plasma pools. Five parameters were measured, FVIII:coagulant activity (FVIII:C), FVIII:Antigen (FVIII:Ag), VWF:Antigen (VWF:Ag), VWF:Ristocetin Cofactor (VWF:RCof), and a new parameter, VWF:collagen binding (VWF:CB). Mean potency estimates for the 4th IS, calculated relative to the 3rd IS, were significantly greater than the mean estimates calculated relative to the fresh normal pools by 15, 14 and 20% respectively for FVIII:C, VWF:Ag and VWF:RCof. These results indicate a drift in the International Unit away from the fresh plasma unit. Partial rectification of this drift was achieved by assigning the mean of the estimates calculated relative to the 3rd IS and the fresh plasma pools, i.e. FVIII:C 0.57 IU/ampoule, VWF:Ag 0.79 IU/ampoule and VWF:RCof 0.73 IU/ampoule. This represents a shift in the IU between the 3rd and 4th IS of 7.5% for FVIII:C, 7% for VWF:Ag and 10% for VWF:RCof. Mean estimates of FVIII:Ag relative to the 3rd IS and the fresh normal pools agreed to give an assigned value of 0.89 IU/ampoule. Excessive inter-laboratory variability and a low number of estimates (n = 6) precluded the assignment of a potency for VWF:CB. The 4th IS Factor VIII/VWF plasma (97/586) was established in October 1998.

International collaborative study to evaluate a recombinant L ferritin preparation as an International Standard

A recombinant L ferritin preparation, lyophilized in ampoules and designated 94/572, was evaluated by 18 laboratories in 9 countries for its suitability as an International Standard (IS). The preparation was assayed in a wide range of in-house and commercial immunoassays against the 2nd IS for ferritin (of spleen origin; 80/578). The immunological reactivity of the recombinant material was similar to that of the 2nd IS for ferritin in the majority of assays and demonstrated adequate stability in accelerated degradation studies. On the basis of the results presented here, the WHO Expert Committee on Biological Standardization established 94/572 as the 3rd IS for ferritin, recombinant.

A Comparison of Heparin Potency Estimates Obtained by Activated Partial Thromboplastin Time and British Pharmacopoeial Assays

Heparin samples from five manufacturers were assayed by the revised British Pharmacopoeia (BP) heparin assay and the results compared with those obtained using the activated partial thromboplastin time (APTT) assay. The United States Pharmacopoeia (USP) reference heparin preparation and the 4th International Standard (IS) for heparin were also assayed by the two methods relative to the 3rd IS. The results obtained by the revised BP assay were in close agreement with those obtained by the APTT assay for all the heparins that were tested. The assays revealed that there is at least a 10% discrepancy between the International Unit for heparin and the USP unit.

Standardization of VIII:C assays: a manufacturer's view.

ABSTRACT: Despite the use of an International Standard to define the unitage of Factor VIII coagulant activity (VIII:C) discrepancies still exist with respect to labelled potencies. In order to examine possible causes of this disagreement concentrates of low, intermediate and high purity, normal plasma (NP) and the 1st International Reference Plasma (IRP) were assayed against the 3rd International Standard (IS) using one‐stage, two‐stage and chromogenic substrate assays.When different concentrates were estimated in relation to the 3rd IS, and NP in relation to the 1st IRP, there was good agreement between the potency estimates obtained by the different assay methods. Consistent results were obtained when VIII:C deficient plasma was used instead of buffer as prediluent. When NP and IRP were assayed against the 3rd IS marked differences between the assay methods were observed when buffer was used as prediluent. This effect was reduced when the 3rd IS was prediluted with VIII:C deficient plasma.Many factors influence the standardization of VIII:C assays, but good agreement between methods can be achieved provided that like preparations are compared under standardized conditions.

Measurement of the Heparin Neutralizing Capacity of Protamine

Several methods of estimating the heparin neutralizing capacity of protamine were investigated for their reliability and practicability. The results from two chemical methods were compared with those from two in vitro biological assays, one of which was the method of the British Pharmacopoeia (1973). An in vivo method using mice was used to assess the accuracy of the in vitro test methods. Three standard heparin preparations were tested against the W.H.O. 1st International Reference Preparation for Protamine. Two of the heparin preparations were of mucosal origin, one of which was the 3rd International Standard, and the third heparin preparation was of lung origin (the 2nd International Standard for Heparin). The mean neutralization values (all methods) of heparin by protamine for a House Reference Preparation and the 3rd and 2nd International Standards for Heparin were 95.8, 109.8 and 89.9 units per mg of Protamine, respectively. All methods read the House Reference Preparation to have a lower value than the 3rd International Standard, which had a higher value than the 2nd International Standard for Heparin. There was a constant relationship between the results of any one method and those of another. The chemical and in vitro biological methods gave results of comparable precision although the latter required a greater degree of technical skill and time to perform.

Anticoagulant activity of heparin: Assay of bovine, human and porcine preparations by amidolytic and clotting methods

The anticoagulant activities of different heparin preparations have been compared using two clotting (APTT and CaTT) and two amidolytic assays. The heparin samples analyzed included two heparin standards, heparin isolated from human mastocytoma tissue, four commercial heparin preparations and two heparin preparations (“high affinity heparin” and “low affinity heparin”) separated by affinity chromatography on immobilized antithrombin III. In each assay system, specific activities were expressed relative to the specific activity of the 3rd International standard (assigned to 100). The specific activities obtained ranged from 3 to 198. In all assay systems “high affinity heparin” had the highest and “low affinity heparin” the lowest specific activity. For each individual heparin preparation the variation in specific activity recorded with the various assay methods was relatively slight. However, the specific activities of the commercial heparins determined in the present study differed considerably from those reported by the manufacturers using pharmacopoeial methods. Exchanging purified antithrombin III with plasma in the amidolytic assay systems caused relatively small changes in specific activities, but suggested that plasma antiheparin activity was more effective in the heparin-antithrombin III-thrombin than heparin-antithrombin III-Xa reaction. In view of the reproducibility and simplicity of amidolytic methods, it is suggested that they are adapted for heparin standardization.