WHO Reference Reagent Research Articles

In vitro Generated Megakaryocytes for the Detection of Human Platelet Antigen-Specific Alloantibodies

Plain Language SummaryThe identification of antibodies to human platelet antigens (HPA) is crucial. The standard test (MAIPA assay) is difficult because it requires fresh platelets from specific donors. This study investigated a new method, monoclonal antibody immobilization of megakaryocyte antigens (MAIMA), using in vitro generated megakaryocytes from CD34+ cells. The assay was tested against WHO reference reagents and patient samples with known anti-HPA antibodies. Results showed that MAIMA successfully detected anti-HPA-1a antibodies and correlated well with MAIPA. However, MAIMA had lower sensitivity for anti-HPA-3a and anti-HPA-5b antibodies. Notably, anti-HPA-5b antibodies were not detectable by MAIMA. In conclusion, in vitro generated megakaryocytes can effectively detect anti-HPA-1a antibodies. However, MAIMA may be less suitable for antibodies against other HPAs, such as HPA-5b. This study highlights the potential of MAIMA as an alternative assay and provides insight into its strengths and limitations in detecting specific anti-HPA antibodies.

Variation in the Measurement of Anti-Müllerian Hormone - What Are the Laboratory Issues?

Anti-Müllerian Hormone (AMH) is a 140 kDa homodimeric glycoprotein consisting of two identical subunits linked by disulphide bonds and is synthesised by the testes and ovaries. Its clinical applications are prediction of ovarian response and gonadotropin dose selection upon in vitro fertilization. In males, AMH is used to investigate sexual developmental disorders and gonadal function. AMH is commonly assayed by enzyme-linked immunosorbent assay or automated immunoassay formats that show variation between methods. This review applies fundamental chemical pathology concepts to explain the observed analytical variation of AMH measurement. We examine the lack of standardisation between AMH assays, the impact of antibody design on variable measurements, consider the analytical detection of AMH isoforms, review analytical interference in AMH measurement, and briefly assess systematic bias between AMH assays. The improved attempt at standardising AMH measurement by the recent approval of a WHO Reference Reagent offers promise for harmonising immunoassay results and establishing consensus medical cut-off points for AMH in disease. Standardisation, however, will need to redress the issue of poor commutability of standard reference material and further assign a standard reference procedure to quantify AMH standard reference material. The improvement of the analytical phase of AMH testing will support harmonised method development and patient care.

The efficacy of ethnic specific blood groups genotyping for routine donor investigation and rare donor identification in Taiwan.

Large-scale single nucleotide variation (SNV)-based blood group genotyping assays have been made available for over a decade. Due to differences in ethnic groups, there is much diversity in clinically important blood group antigens and genetic variants. Here, we developed a robust matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF)-based blood group genotyping method on MassARRAY system. A total of 1428 donors were enrolled into three groups: (a) reagent red cell donors; (b) rare donor or common antigen-negative donors; and (c) group O, R1 R1 /R2 R2 donors. Forty-two SNVs were designed for determining nine blood groups, with X/Y chromosome in two multiplex reactions, on MassARRAY 96-well format system. Further targeted sequence analyses were performed by Sanger sequencing. WHO reference reagent (NIBSC code: 11/214) was tested for concordance with the provided genotype results. Among the donors, concordance rate was over 99%. Alleles of important phenotypes such as Mi(a+), Di(a+), and Asian-type DEL and alleles of rare blood groups such as Fy(a-), Jk(a-b-) and s- were screened. Three types of discrepancies were found. Serologically, the 'N' antigen was expressed on genetically MM with GYP*Mur red blood cells and caused genuine discrepancies (9.5%). Genetically, allele dropout (ADO) was caused by rare SNV in the primer for Ss genotype (2.1%) and partial insertion of RHD genes (0.9%) led to difficulties in predicting phenotypes. Hemo panel module and MassARRAY System in 96-well format showed good performance in terms of large-scale blood group genotyping and phenotype predictions. Implementation of this method is effective for routine blood group genotype screening of donors.

Complete Genome Sequence, Genome Stability and Phylogeny of the Vaccine Strain Mycobacterium bovis BCG SL222 Sofia.

Mycobacterium bovis bacillus Calmette–Guérin (BCG) is the only live attenuated vaccine available against tuberculosis. The first BCG vaccination was done exactly 100 years ago, in 1921. The BCG vaccine strains used worldwide represent a family of daughter sub-strains with distinct genotypic characteristics. BCG SL222 Sofia is a seed lot sub-strain descending from the Russian BCG-I (seed lot 374a) strain and has been used for vaccine production in Bulgaria since 1972. Here, we report the assembled circular genome sequence of Mycobacterium bovis BCG SL222 Sofia and phylogeny analysis with the most closely related BCG sub-strains. The full circular genome of BCG SL222 Sofia had a length of 4,370,706 bp with an average GC content of 65.60%. After 49 years of in vitro evolution in a freeze-dried condition, we identified four SNP mutations as compared to the reference BCG-I (Russia-368) sequence. BCG vaccination is of central importance for the TB elimination programs in many countries. Since 1991, almost 40 million vaccine doses of the BCG SL222 Sofia have been distributed annually through the United Nations Children’s Fund (UNICEF) and the Pan American Health Organization (PAHO) to approximately 120 countries. The availability of the complete reference genome sequence for M. bovis BCG SL222 Sofia, a WHO reference reagent for the Russian BCG-I sub-strain, will facilitate the identity assurance of the genomic stability, will contribute to more consistent manufacturing, and has an important value in standardization and differentiation of sub-strains used in vaccine production. We propose to rename the sub-strain BCG SL222 Sofia to BCG-Sofia for practical and common use.

Application of lyophilised human platelets for antibody detection in solid phase red cell adherence assay

Antibodies against human platelets cause a variety of thrombocytopenic disorders, which lead to potentially fatal haemorrhage. Therefore, their prompt detection is mandatory for successful patient treatment. Solid phase red cell adherence (SPRCA) assay allows for platelet antibody detection widely. However, preparation of fresh platelets with HLA-I and human platelet antigens (HPA)1–5,15 genotyped as target cells is inconvenient and fresh platelets have a short shelf life. In this study, the lyophilised human platelets for antibody detection in SPRCA were prepared. Firstly, platelets were resuspended in lyophilisation buffer and freeze-dried. Then the characteristics of lyophilised platelet were analysed. Rehydrated platelets were recovered with a mean rate of 80.91% ± 2.87%, and still retained spherical morphology. Indirect flow cytometry showed that glycoproteins IIb/IIIa, Ia/IIa, Ib/IX, IV, CD109, and HLA class I were present on the surface of the lyophilised platelets at a comparable level to that of fresh platelets. The consistent results obtained with WHO reference reagents containing anti-HPA-1a, anti-HPA-3a, and anti-HPA-5b, as well as clinical samples from the same donors containing anti-HLA antibodies when reacting with lyophilised versus fresh platelets confirmed good antigenicity preservation of platelets after freeze-drying. Further investigation showed that the lyophilised platelets could be stored at 2–8 °C for up to 14 months and the reconstituted suspension was stable for 48 h. Therefore, lyophilised platelets can be a convenient alternative to fresh platelets to use for anti-platelet antibody detection in SPRCA tests.

Establishment of a WHO Reference Reagent for anti-Mullerian hormone

BackgroundThere is a need for a reference material to support the development and ensure the quality of immunoassays for human AMH. A batch of ampoules, coded 16/190, containing lyophilised recombinant AMH was evaluated in a WHO Collaborative Study. The aims of the study were to determine the AMH content in terms of the calibration of each immunoassay method, to predict long-term stability and to assess the suitability of the preparation to calibrate AMH immunoassays.MethodsStudy participants were asked to report the AMH content of specific dilutions of coded ampoules of 16/190 and a comparator preparation containing approximately half the AMH content. In each assay, participants also reported the AMH content of 22 patient samples to assess commutability. A robust all-laboratory geometric mean of the content estimates was determined using the laboratory geometric mean estimates. Commutability was assessed using a difference in bias approach. Stability was predicted by the measurement of thermally accelerated degradation samples.ResultsSeven laboratories performed twenty-one immunoassay method-platform combinations, sixteen of which provided data which met the validity criteria, giving a consensus geometric mean estimate of AMH content of 511 ng/ampoule (95% CI, 426–612, n = 16, GCV 42%) and a robust geometric mean of 489 ng/ampoule. By contrast, the GCV% for the all-laboratory geometric mean of the relative content estimates for the comparator sample to 16/190 was 12%. Commutability was assessed using 20 of the 22 representative patient samples. Of the valid assays, 16/190 was within the limits of acceptable commutability for 6 methods, partially commutable for a further 3 methods and non-commutable when measured by 7 methods. The preparation was predicted to be highly stable when stored at − 20 °C.ConclusionThe majority of methods met the validity criteria. Content estimates showed a high between-method variability, yet assays exhibited a similar proportionality of response as demonstrated using the comparator sample. 16/190 was commutable in some but not all methods. On the basis of these results, it was agreed by the WHO Expert Committee on Biological Standardization to establish 16/190 as a WHO Reference Reagent for AMH with a content defined by consensus immunoassay of 489 ng/ampoule.

A WHO Reference Reagent for lupus (anti-dsDNA) antibodies: international collaborative study to evaluate a candidate preparation

IntroductionAntibodies against double-stranded DNA (anti-dsDNA) are a specific biomarker for systemic lupus erythematosus (SLE). The first WHO International Standard (IS) for anti-dsDNA (established in 1985), which was used to assign...

Comparison of pellicle and shake flask-grown BCG strains by quality control assays and protection studies

A global BCG vaccine shortage began in 2013 which impacted availability for infant vaccinations, as well as preclinical studies and clinical trials of new TB vaccines. Stakeholders met in 2015 at McGill University in Montreal to discuss the shortage and potential mitigation strategies. Manufacturing BCG through a more tractable liquid fermentation process instead of the traditional pellicle growth method was considered a potentially viable strategy. This pilot program compared pellicle-grown and shake flask-grown BCG strains (as a first step towards modeling fermenter-produced BCG vaccine) in selected quality control assays, as well as mouse and guinea pig protection studies. Conventional pellicle-grown, lyophilized BCG WHO Reference Reagents (Danish, Moreau, Russian, Tokyo strains) were obtained from the National Institute for Biological Standards and Control (NIBSC), UK. Strains were grown in shake flasks and glycerol stocks prepared. Shake flask-grown BCG culture preparations generally met the requirements of quality control testing at NIBSC. In mouse and guinea pig protection studies there were no significant differences in lung colony forming units (CFUs) between shake flask-grown and pellicle-grown preparations, with the exception of BCG Russian, where the shake flask-grown preparation protected better in mice (P = 0.0042), but the pellicle-grown preparation protected better in guinea pigs (P = 0.0015). Producing BCG vaccines by a more tractable liquid growth process could be a viable solution to the global BCG shortage.

Optimization of incubation conditions of Plasmodium falciparum antibody multiplex assays to measure IgG, IgG1\u20134, IgM and IgE using standard and customized reference pools for sero-epidemiological and vaccine studies

BackgroundThe quantitative suspension array technology (qSAT) is a useful platform for malaria immune marker discovery. However, a major challenge for large sero-epidemiological and malaria vaccine studies is the comparability across laboratories, which requires the access to standardized control reagents for assay optimization, to monitor performance and improve reproducibility. Here, the Plasmodium falciparum antibody reactivities of the newly available WHO reference reagent for anti-malaria human plasma (10/198) and of additional customized positive controls were examined with seven in-house qSAT multiplex assays measuring IgG, IgG1–4 subclasses, IgM and IgE against a panel of 40 antigens. The different positive controls were tested at different incubation times and temperatures (4 °C overnight, 37 °C 2 h, room temperature 1 h) to select the optimal conditions.ResultsOverall, the WHO reference reagent had low IgG2, IgG4, IgM and IgE, and also low anti-CSP antibody levels, thus this reagent was enriched with plasmas from RTS,S-vaccinated volunteers to be used as standard for CSP-based vaccine studies. For the IgM assay, another customized plasma pool prepared with samples from malaria primo-infected adults with adequate IgM levels proved to be more adequate as a positive control. The range and magnitude of IgG and IgG1–4 responses were highest when the WHO reference reagent was incubated with antigen-coupled beads at 4 °C overnight. IgG levels measured in the negative control did not vary between incubations at 37 °C 2 h and 4 °C overnight, indicating no difference in unspecific binding.ConclusionsWith this study, the immunogenicity profile of the WHO reference reagent, including seven immunoglobulin isotypes and subclasses, and more P. falciparum antigens, also those included in the leading RTS,S malaria vaccine, was better characterized. Overall, incubation of samples at 4 °C overnight rendered the best performance for antibody measurements against the antigens tested. Although the WHO reference reagent performed well to measure IgG to the majority of the common P. falciparum blood stage antigens tested, customized pools may need to be used as positive controls depending on the antigens (e.g. pre-erythrocytic proteins of low natural immunogenicity) and isotypes/subclasses (e.g. IgM) under study.

Establishment of the 1st WHO International Standard for anti-EV71 serum (Human)

Enterovirus A71 (EV71) is the major causative agent of severe and fatal hand, foot and mouth disease. There is plenty of evidence that EV71 has circulated widely in the Western Pacific Region for the last twenty years. Vaccines against EV71 are already available or under development. A collaborative study to establish the 1st WHO International Standard for anti-EV71 serum (Human) was conducted to ensure that methods used to measure the serum neutralizing activity or antibody levels against EV71 are accurate, sensitive and reproducible. Two candidate samples as well as a third candidate reference containing low anti-EV71 antibody titre were produced from plasma samples donated by healthy individuals. All three serum samples exhibited good levels of neutralizing antibodies against a wide range of EV71 strains of various genotypes. The study showed that between laboratory variations in neutralization titres were significantly reduced when values were expressed relative to those of either of the two candidate sera. Sample 14/140 was established as the WHO 1st International Standard for anti-EV71 serum (human), 14/138 as its potential replacement and 13/238 as a WHO Reference Reagent, with assigned unitage of 1,000, 1090 and 300 International Units (IU) of anti-EV71 neutralizing antibodies per ampoule, respectively.

Bioluminescence Methods for the Rapid Assay of the Specific Activity of Lyophilized BCG Vaccine

The specific activity of a lyophilized BCG vaccine (CFU/mg biomass) is one of the most important characteristics of this product. The classical microbiological method of the BCG vaccine activity assay is very time-consuming and gives results only in 4-5 weeks. The bioluminescent determination of specific activity based on the use of the firefly luciferase-luciferin system makes it possible to greatly shorten the duration of the assay. Since the 1970’s, the ATP method has been successively applied for studying the specific activity of different BCG strains. In 2008, the bioluminescent ATP method was proposed and evaluated for analyzing lyophilized BCG vaccine preparations. The duration of analysis decreased from several weeks to 2 days. This method was used in the International collaborative study to evaluate and establish WHO Reference Reagents for BCG vaccine. In 2016, a new protocol was published describing the use of the ATP method to assay the specific activity of the lyophilized BCG vaccines. The duration of the assay decreased to 2 h. A good correlation between the CFU value and the intracellular ATP content was shown.

The establishment of sub-strain specific WHO Reference Reagents for BCG vaccine

As the latest addition to the sub-strain specific WHO Reference Reagents of BCG vaccine, an international collaborative study was completed to evaluate the suitability of a candidate BCG Moreau-RJ sub-strain as a WHO Reference Reagent of BCG vaccine. This follows the recent replacement of the WHO 1st International Reference Preparation for BCG vaccine, by three sub-strain specific WHO Reference Reagents of BCG vaccine (Danish 1331, Tokyo 172-1 and Russian BCG-I) in order to complete the coverage of most predominant sub-strains used for BCG vaccine production and distribution for use worldwide. The study used cultural viable count and modified ATP assays to quantify the preparation and multiplex PCR to confirm the identity of the sub-strain. The establishment of this WHO Reference Reagent of BCG vaccine of Moreau-RJ sub-strain was approved by the WHO Expert Committee on Biological Standardization meeting in October 2012. This preparation is available for distribution by NIBSC-MHRA, UK. The data from real-time stability monitoring demonstrated that these Reference Reagents of BCG vaccine are very stable in storage condition at −20°C. They serve as the valuable source of BCG Reference Reagents for use as comparators (1) for viability assays (such as cultural viable count and modified ATP assays); (2) for in vivo assays (such as the absence of virulent mycobacteria, dermal reactivity and protection assays) in the evaluation of candidate TB vaccines in non-clinical models; (3) for identity assays using molecular biology techniques.

Immunogenicity of An Interferon-β1a Product

In order to determine whether Blastoferon®, a biosimilar interferon (IFN)- beta 1a formulation, shares epitopes with other known IFN-beta products, a series of neutralization bioassays were performed with a set of well-characterized anti-IFN- beta monoclonal antibodies and human sera (World Health Organization Reference Reagents). The bioassay was the interferon-induced inhibition of virus cytopathic effect on human cells in culture (EMC virus and A-549 cells). Computer-calculated results were reported as Tenfold Reduction Units (TRU)/ml. To further assess Blastoferon® immunogenicity, in vivo production of anti-IFN beta antibodies was determined in sera of patients included in the pharmacovigilance plan of Blastoferon® by the level of IFN- beta 1a binding antibodies (by enzyme immunoassay -EIA) and neutralizing antibodies (in the Wish-VSV system). The highly characterized neutralizing monoclonal antibodies A1 and A5 that bind to specific regions of the IFN- beta molecule reacted positively with the three beta 1a IFNs: Blastoferon®, Rebif®, and the IFN- beta WHO Second International Standard 00/572. As expected, the non-neutralizing monoclonal antibodies B4 and B7 did not neutralize any of the IFN- beta preparations. The commercially available monoclonal antibody B-02 reacted essentially equally with Rebif® and Blastoferon®. The WHO Reference Reagent human serum anti-IFN- beta polyclonal antibody neutralized all the IFN- beta products, whereas the WHO Reference Reagent human serum anti-IFN-alpha polyclonal antibody G037-501-572 appropriately failed to react with any of the IFN- beta products. On the basis of in vitro reactivity with known, well-characterized monoclonal and polyclonal antibody preparations, Blastoferon® shares immunological determinants with other human interferon- beta products, especially IFN- beta 1a. In vivo antibodies were detected by EIA in 72.9% of 37 chronically treated multiple sclerosis patients, whereas neutralizing antibodies were found in 8.1% of them. Blastoferon® appears to have immunological characteristics comparable to other IFN- beta 1a products.

Report of an International collaborative study to establish the first WHO reference reagents for BCG vaccines of three different sub-strains

The WHO First International Reference Preparation for BCG vaccine is over forty years old and is no longer available for distribution due to stock depletion and its significant loss of viability. International consultations identified a demand for replacement with sub-strain specific BCG preparations. An International collaborative study was carried out to evaluate three candidates for WHO Reference Reagent for BCG vaccine of Danish 1331, Russian BCG-I and Tokyo 172-1 sub-strains. These candidates were quantified for viability using both cultural viable count and modified ATP assays. The proposal for the establishment of these First WHO Reference Reagents for BCG vaccines was discussed in the WHO Expert Committee on Biological Standardization meeting, October 2009.

WHO international cytokine standards and reference preparations

Abstract How to access WHO International Standards and Reference Reagents for cytokines and growth factors.

International Comparison of C-Peptide Measurements

C-peptide measurement has been widely used as a marker of insulin secretion in patients with diabetes. We assessed the comparability of C-peptide results obtained with different methods and by different laboratories and determined whether C-peptide results could be harmonized by normalization with a WHO reference reagent or with plasma. We sent 16 different heparin plasma samples to 15 laboratories in 7 countries. The samples were analyzed with 10 different assay methods. A WHO C-peptide standard was also sent to each laboratory and used to determine the feasibility of normalizing results. To assess the impact of calibrator matrix on the comparability of results, we also used the mean results of all laboratories for 4 of the samples to normalize the remaining sample results. Between-laboratory variability increased with increasing C-peptide concentrations. Normalization of results with WHO reference reagents did not improve comparability, but normalization with samples significantly improved comparability among laboratories and methods. The 95% confidence interval estimate for the SD for the lab/method effect (0.0-0.061) using sample-normalized values did not overlap with the 95% CI estimate with the raw data (0.090-0.225). C-peptide results generated by different methods and different laboratories do not always agree, especially at higher concentrations of C-peptide. These data support the concept of using a single laboratory for multisite studies and support efforts to harmonize C-peptide measurements by use of calibrators prepared in the sample matrix.

Glycosylation patterns of human chorionic gonadotropin revealed by liquid chromatography‐mass spectrometry and bioinformatics

Due to their extensive structural heterogeneity, the elucidation of glycosylation patterns in glycoproteins such as the subunits of human chorionic gonadotropin (hCG), hCG-alpha, and hCG-beta, remains one of the most challenging problems in the proteomic analysis of post-translational modifications. In consequence, glycosylation is usually studied after decomposition of the intact proteins to the proteolytic peptide level. However, by this approach all information about the combination of the different glycopeptides in the intact protein is lost. In this study we have, therefore, attempted to combine the results of glycan identification after tryptic digestion with molecular mass measurements on the native starting material of the new first WHO Reference Reagents (RR) for hCG-alpha (99/720) and hCG-beta (99/650). Despite the extremely high number of possible combinations of the glycans identified in the tryptic peptides by HPLC-MS (>1000 for hCG-alpha and >10 000 for hCG-beta), the mass spectra of intact hCG-alpha and hCG-beta revealed only a limited number of glycoforms present in hCG preparations from pools of pregnancy urines. Peak annotations for hCG-alpha were performed with the help of a bioinformatic algorithm that generated a database containing all possible modifications of the proteins, including modifications possibly introduced during sample preparation such as oxidation or truncation, for subsequent searches for combinations fitting the mass difference between the polypeptide backbone and the measured molecular masses. Fourteen different glycoforms of hCG-alpha, containing biantennary, partly sialylized hybrid-type glycans, including methionine-oxidized and N-terminally truncated forms, were identified. Mass spectra of high quality were also obtained for hCG-beta, however, a database search mass accuracy of +/-5 Da was insufficient to unambiguously assign the possible combinations of post-translational modifications. In summary, mass spectrometric fingerprints of intact molecules were shown to be highly useful for the characterization of glycosylation patterns of different hCG preparations such as the new first WHO RR for immunoassays and could be the first step in establishing biophysical reference methods for hCG and related molecules.

Establishment, Value Assignment, and Characterization of New WHO Reference Reagents for Six Molecular Forms of Human Chorionic Gonadotropin

The International Federation of Clinical Chemistry and Laboratory Medicine (IFCC) established a Working Group to investigate means of improving the comparability of immunoassays for human chorionic gonadotropin (hCG), which was selected as a prototype glycoprotein analyte. The Working Group identified development of unambiguous nomenclature and production of new highly purified International Reference Reagents calibrated in substance concentrations as its primary objectives. Preparations of intact hCG, nicked hCG, hCG beta-subunit, nicked hCG beta-subunit, hCG alpha-subunit, and hCG beta-core fragment were purified from a crude urinary hCG preparation, ampouled, lyophilized, and assigned values in substance concentrations (mol/L). Value assignment and accelerated degradation studies were carried out in accordance with WHO protocols for International Reference Reagents. The ampouled standards were assigned final values based on the recovery of immunoreactive material after reconstitution. The degradation studies showed that the standards were highly stable. The nomenclature of hCG-related molecules and immunoassays has been adopted by the IFCC, and the standards prepared and characterized by the Working Group have been formally adopted by the WHO as the First International Reference Reagents for six hCG-related molecules. These developments will enable better understanding of what assays for hCG measure and should ultimately help to improve the clinical application of these assays.

Collaborative studies to establish the first WHO Reference Reagent for detection of human antibody against human platelet antigen-5b.

This report describes the production of a freeze-dried preparation of pooled human plasma, coded 99/666, containing immunoglobulin G (IgG) antibodies against human platelet antigen 5b (HPA-5b). The material is intended for use as a minimum sensitivity reagent in the assays currently used for detection of antibodies to HPA-5b. Laboratories can use it to assess the sensitivity of their 'in-house' assays for antibodies to HPA-5b and to calibrate local controls for routine use in each batch of tests. Two collaborative studies demonstrated that the two candidate materials contained antibodies to HPA-5b and that there were no other HPA or human leucocyte antigen (HLA) antibodies which might confuse the detection of antibodies to HPA-5b. The two samples were pooled and freeze-dried in 1-ml ampoules. The minimum dilution of the antibody against 5b required to yield a positive result was determined, by two international collaborative studies involving a total of 49 laboratories in 26 countries, to be 1 in 2.

Preparation and characterization of new WHO reference reagents for human chorionic gonadotropin and metabolites.

The currently used standards for human chorionic gonadotropin (hCG) and its alpha and beta subunits (hCGalpha and hCGbeta) contain substantial amounts of contaminating variants of hCG and other impurities. Furthermore, some partially degraded forms of hCG and its subunits have become of potential clinical importance, e.g., "nicked" forms of hCG (hCGn) and hCGbeta (hCGbetan), which contain cuts in the peptide backbone between amino acids 44-45 or 47-48 in hCGbeta, and a fragment of hCGbeta (hCGbetacf) consisting of amino acids 6-40 and 55-92 bound together by disulfide bridges. The IFCC appointed a working group with the aim of preparing new standards for hCG and related substances to improve standardization of their immunoassays. Large amounts of hCG and its subunits as well as of hCGn, hCGbetan, and hCGbetacf were prepared by previously developed purification methods in combination with hydrophobic interaction chromatography and reversed-phase HPLC. Each preparation was characterized on the basis of amino acid and sequence analyses, carbohydrate composition, and electrophoretic patterns. Immunoassays for relevant contaminating proteins were also performed. The major preparations were homogeneous and free of contaminating proteins. Concentrations of the final preparations were determined by amino acid analysis. Calibrated in substance concentrations (mol/L) based on amino acid analyses, these preparations will facilitate improved standardization of immunoassays for hCG and its metabolites. The six preparations have now been established by the WHO as new 1st Reference Reagents for immunoassays with the following codes: hCG 99/688, hCGbeta 99/650, hCGalpha 99/720, hCGn 99/642, hCGbetan 99/692, and hCGbetacf 99/708. In contrast to the 3rd International Standard (75/537), the clinically most important Reference Reagent for hCG (99/688) contains no hCGn and negligible amounts of free subunits.