The immuassay handbook parte36

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The immuassay handbook parte36

  1. 1. 315© 2013 David G. Wild. Published by Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/B978-0-08-097037-0.00021-X As in all forms of measurement there is a need for immunoassay standardization. Standardization is the pro- cess of ensuring that all methods for determining the con- centration of a particular analyte give the same results. Calibration is the process of assigning values to unknown samples using a standard. Immunoassays do not provide direct measurements (unlike a meter rule for example). They estimate analyte concentration in unknown samples by comparing signal strength (originating from a labeled reagent) with that from similarly treated standard samples. However, for most of the analytes determined by immu- noassay, there are no reference methods with which to calibrate standards. This presents a dilemma that is far from resolved for many immunoassay analytes. The importance of standardization is often underesti- mated. It is usual practice to state the reference interval (see CLINICAL CONCEPTS) for a particular method alongside patient results, but once a reference interval becomes lodged in the memory it can be difficult to avoid it being used subconsciously to interpret results. This can have seri- ous implications for patient diagnosis if a laboratory changes method and the reference interval is significantly different. Lack of standardization hinders communication and sci- entific progress. For example it reduces the value of com- prehensive age and sex-related reference intervals in the scientific literature, which could otherwise be used to improve the diagnostic capability of different manufactur- ers’ tests for the same analyte. There are also knock-on effects. For example, some publications stated that a lutein- izing hormone: follicle stimulating hormone (LH:FSH) ratio of greater than three is diagnostic of polycystic ovar- ian syndrome. However, subsequent immunometric assays for LH gave much lower values than the methods that had been used when this ratio was established, making the use of a value of three as a diagnostic cut-off inappropriate. Standardization requires: G Value assignment in meaningful units. G A standard that is identical to the analyte in the test samples. G Absence of interference from the test sample matrix. G A reference method. G Demonstration of inter-method agreement. As will be seen in this chapter, these criteria are rarely met in the immunoassay field and standardization is beset with problems. Diligent efforts by many different organizations have led to a form of consensus, but there have had to be some compromises, caused primarily by the limitations of immunoassay technology and the heterogeneity of patient samples. However there is a great scope for improved stan- dardization and this is a fertile area for development. Dur- ing a course of treatment, individual patients are more likely than ever before to have tests carried out for the same analyte by different methods (e.g. point-of-care and labora- tory analyzer), and to have their results checked against ref- erence intervals that were set elsewhere, and perhaps not using the same method. Therefore there is now an even greater need for standardization, and this field is still one of the greatest challenges faced by immunoassay scientists. Standardization THE ROLE OF EXTERNAL QUALITY ASSESSMENT (PROFICIENCY TESTING) SCHEMES Historically, the first step in achieving the standardization of different immunoassays for the same analyte has been the initiation of a regular external quality assessment (pro- ficiency testing) scheme. Pooled samples are sent to a number of laboratories and the results are analyzed by the scheme coordinator. Often the first results show a wide variation. However it has been shown for many analytes that the strategy of feeding back biases from the all-laboratory trimmed mean (ALTM) to scheme participants and manu- facturers leads eventually to a reduction in the overall vari- ation. Methods that have consistent bias from other methods are clearly highlighted, and laboratories that are not in consensus with their peer group are made aware of their relative position. Some scheme organizers send out spiked samples to estimate the recovery and this can help to pinpoint fundamental problems with individual methods. As collaborative efforts to achieve standardization occur, the improvement can be tracked in the scheme and aber- rant methods become more clearly identifiable. Studies have shown that the ALTM tends to be close to the ‘true’ concentration, but this cannot be taken for granted in the absence of a reference method, although recovery experi- ments may help to identify inappropriately standardized methods. It is the responsibility of participants in external quality assessment schemes to notify manufacturers of significant bias of their method from the ALTM. INTERNATIONAL STANDARDS Ultimately, to achieve agreement between different methods, a single recognized standard is needed. To achieve widespread credibility, it is best prepared under the overall management of an international body. Some analytes are available in a highly purified form, e.g. corti- sol, available from the NIST, Washington DC, USA, at 98.9 ± 0.2% purity. However, most International Standards for immunoassay analytes are prepared by the Standardization and Calibration David Wild (david@davidwild.net) Chris Sheehan C H A P T E R 3.5
  2. 2. 316 The Immunoassay Handbook National Institute for Biological Standards and Control in the UK (www.nibsc.ac.uk). Preparation of International Standards is a specialized activity and validation of each step is required, with sup- port from several independent laboratories. One of the frequent problems of standard preparation is heterogene- ity. Many immunoassay analytes are proteins with varying degrees of glycosylation (attachment of sugar residues). Some of this glycosylation needs to be retained if the stan- dard is to be of use. Gentle methods of purification are used to try and preserve the structural integrity of the ana- lyte. The starting material must also be from a reliable source and, if the analyte is a protein, of human origin. If the protein is extracted from organs, an organ is selected with a protein structure as near to that found in the blood as possible. The purified protein is mixed with an inert carrier com- pound, otherwise the amount would be too small to be vis- ible. Then the solution is divided into aliquots in glass ampoules, freeze-dried, and the ampoules are sealed under nitrogen. After demonstrating reproducibility across the ampoules (better than ±0.25%) and ensuring a low residual moisture content has been achieved, samples are sent to laboratories for independent estimates of concentration and to demon- strate that the standard analyte in the preparation behaves similarly to the naturally occurring analyte in human blood samples. To be designated an International Standard (IS), the preparation has to be authenticated by the Expert Com- mittee on Biological Standardization (ECBS) of the World Health Organization (WHO). An IS is a preparation to which an International Unit (IU) has been assigned on the basis of an international collaborative study involving several assay systems in different laboratories. The term ‘IRP’ (International Reference Preparation) is no longer used for new materials. It used to be assigned to prepara- tions that did not meet the demanding criteria for an IS but were useful for method to method standardization. The designation of IS status is sometimes portrayed as the end of the road for standardization. It is, without doubt, a significant achievement to achieve worldwide agreement on a common standard, but International Units (IU) are still arbitrary units. To some extent, this is a ves- tige from enzyme determinations, which were expressed in terms of substrate conversion activity units, and from bioassays, which detected hormones in samples through their biological activity. Where protein concentrations are not stated in terms of IU, they are sometimes presented in terms of mass, e.g. ng/L. The limitations of arbitrary and mass units were illustrated by the unitage of the Interna- tional Standard for hCG (IS 75/537), and the Interna- tional Reference Preparations for the α subunit (IRP 75/569) and β subunit (IRP 75/551) of hCG. The IS value assignment was based on bioassay units, and the subunit IRPs were based on mass, so they were not easily compa- rable, except by using crude conversion factors. (Note that the latest versions of these standards are: 75/589 (intact hCG), 99/720 (α subunit) and 99/650 (β subunit), and there is a special 1st WHO Reference Reagent (99/688) for intact hCG for immunoassays). At first sight, mass concentration units might seem to be ideal as units of measurement for immunoassay analytes but they also have limitations, because of the variation in the molecular weights of different analytes. For example the presence of the same concentration of β subunit molecules of hCG in one sample as intact protein in another sample would result in a mass concentration that is approximately half as much, because of the lower molecular weight. Ultimately immunoassay standardization should be in more meaning- ful terms, and it is to be hoped that one day we will see concentrations of proteins expressed in molar concentra- tion. To achieve this, much work needs to be done on the characterization of the proteins concerned. Progress has been made for insulin and adrenocorticotrophic hormone (ACTH) and their concentrations are often expressed as moles per liter. The problems associated with assigning molar concen- trations to existing International Standards are very sig- nificant. A designation of a molar concentration to no more than two significant figures by one laboratory could soon be followed by another announcing a different esti- mate, and this would not further the cause of standardiza- tion at all. The problems associated with assigning molar concentrations to heterogeneous analytes are particularly complex and it has even been argued that this goal is scien- tifically invalid. However a pragmatic approach to these difficulties is emerging, as demonstrated by the current IS for FSH for immunoassay (92/510), which is a recombi- nant DNA preparation, giving it a defined structure, and the standard is accompanied by a statement of the approxi- mate content in moles based on uv absorbance measure- ments and the theoretical extinction coefficient of the peptide chain. The IS designation has helped to resolve potential dif- ferences between European and US attempts to standard- ize locally. Many organizations are active in the area of standardization, including the Bureau Communautaire de Référence (BCR) in Brussels, now known as the IRMM (Institute for Reference Materials and Measurement), and in the US the Centers for Disease Control (CDC), Col- lege of American Pathologists (CAP), National Commit- tee for Clinical Laboratory Standardization (NCCLS), which is now known as Clinical Laboratory and Standards Institute (CLSI), the National Institute of Standards and Technology (NIST) and the National Institute of Health (NIH), and these organizations have subtle differences in approach. The International Federation of Clinical Chem- ists (IFCC) has been active in encouraging standardization worldwide. Long-term stability of International Standards is tested using parallel storage at a number of different tempera- tures. Some indication can be given using an Arrhenius plot of activity vs time for each temperature (accelerated degradation testing), and there is a rough rule-of-thumb that shelf-life doubles for each 10°C reduction in temper- ature. A pragmatic approach that has been adopted by the WHO is to store samples of the standards at −20°C and −80°C. If no differences are observed in activity, it is a safe assumption that both stocks are secure and have not lost integrity. In this way a stable international standardization for insulin has been maintained for over 70 years, through several generations of standard, each calibrated from its predecessor.
  3. 3. 317CHAPTER 3.5 Standardization and Calibration DEFINITIVE AND REFERENCE METHODS In the field of clinical chemistry certain well-established methods have been chosen as definitive or reference methods ((NCCLS document NRSCL12-P; EP15-A2 User Verification of Performance for Precision and True- ness; Approved Guideline – Second Edition (www.clsi. org)). A definitive method is the most desirable. It will have been thoroughly investigated and evaluated for sources of inaccuracy, including non-specificity, and uses accurate primary reference materials. A definitive method is considered to be accurate and precise, and suitable for calibration of all methods and standards in the US. A refer- ence method may be a subsidiary method, calibrated from the definitive method, used locally to calibrate field meth- ods and reference materials, or it can be a future candidate for definitive method status, still undergoing evaluation. Reference methods have been thoroughly investigated, and the methods have been very concisely documented to allow repeatability. For some analytes, reference methods are considered the highest category, because experts con- sider that a definitive method is unlikely to become available. An immunoassay for one analyte, digoxin, has been sub- mitted as a candidate reference method. Yet, for many analytes, it is difficult to prove that one method is more accurate than another, as immunoassay is an indirect mea- surement technique, and depends on the use of standards with predetermined or arbitrary values assigned to them, and an ill-defined biological reagent: the antibody. Immu- noassays do not measure defined physical or chemical parameters, such as moles or grams, directly. However, they have the advantage of a level of analytical sensitivity that is unmatched by technologies that would otherwise be preferred for standardization. For a few immunoassay analytes, independent refer- ence methods do exist. ID-GCMS (isotope dilution-gas chromatography mass spectrometry) is suitable as a refer- ence method for cortisol. It involves the separation of cortisol by gas–liquid chromatography, specific detection by mass spectrometry, and determination of the recovery of a heavy isotope label added to the sample. A reference preparation of cortisol was prepared with a high purity and authenticated by international agreement. Several specialist laboratories around the world that had devel- oped ID-GCMS methods validated them independently, using the reference preparation. As a final test, patient samples were submitted to each reference laboratory and their estimates of concentration were confirmed to agree within tight tolerances. Only then was the method assigned the status of ‘reference method’. For an account of the use of ID-GCMS reference methods and other aspects of the standardization of cortisol assays see Gosling et al. (1993). The availability of the reference method for cortisol enabled the UK National External Quality Assessment Scheme (UK NEQAS) to lead the standardization process across the industry, by providing to scheme participants data on their bias on a range of control samples over a period of many months. Serum pools containing cortisol were calibrated using ID-GCMS by a reference labora- tory in Cardiff and sent to testing laboratories around the UK. Several immunoassay methods were found to be biased with respect to ID-GCMS, some by as much as 25%. One method was biased by 20%, even though the assay calibrators had been calibrated using ID-GCMS at the same reference laboratory in Cardiff. This large dis- crepancy was because the calibrators were made using charcoal-stripped human serum as a base-matrix and, therefore, did not closely mimic patient samples. Although the calibrators were correctly assigned cortisol values, the immunoassay did not behave identically when producing signal from patient samples (This is a classic example of a matrix effect). Over a period of time, the various com- mercial immunoassay methods for cortisol were recali- brated by the manufacturers to align them with ID-GCMS. This exercise had the beneficial effect of reducing varia- tion in the UK, and linking the immunoassay methods to a reference method, improving accuracy. Similar cam- paigns have been successful in Germany for steroids such as estradiol. It is sometimes erroneously assumed that all ID-GCMS methods are reference methods. However, they can only be designated as reference methods when several highly controlled laboratories validate their methods internally, to a high standard, then agree with each other within close limits in an international study. Unfortunately, ID-GCMS is only suitable for small homogeneous molecules such as steroids. There are few reference methods for proteins and, to make matters more difficult, many proteins, especially glycoproteins, are het- erogeneous (i.e. they exist in different isoforms). In order to calibrate an immunoassay in the absence of a reference method, it is necessary to use pure analyte or a purified preparation that has been agreed to as an International Standard. OTHER REFERENCE MATERIALS International Standards are supplied as highly purified protein extracts, without contamination by serum or plasma. Calibration of immunoassays using these materi- als as primary standards does not guarantee that patient samples will give the same results in different assays. For this reason international reference materials provided in a human serum based matrix have been prepared. These include cortisol and progesterone certified reference materials (CRM) from IRMM, the Serum Protein Stan- dard (with values for 14 analytes), and the Apolipoprotein Standard. HETEROGENEITY OF STANDARD MATERIAL Many analytes, especially glycoproteins, are naturally heterogeneous. The sources of heterogeneity include glycosylation, sialylation, polymerization, aggregation and decomposition. Different levels of prohormones (e.g. ACTH), subunits (e.g. α- and β-hCG), fragments (e.g. PTH), complexes (e.g. PSA) and subtypes (e.g. CEA) create additional diversity that may affect one immunoassay method more than another. Natural varia- tion between individuals occurs, and certain forms can increase or decrease with different physiological states, sex or age.
  4. 4. 318 The Immunoassay Handbook It is important to use purified materials for calibration purposes but the purification process may further alter the structure or properties of the analyte. Because the IS rep- resents only one sample of a heterogeneous analyte, meth- ods standardized against the same IS may vary in the values they provide for a given series of patient samples. This is because each immunoassay method may have a unique set of antibodies that recognize specific epitopes on the ana- lyte. Dual monoclonal antibody assays tend to be more selective than competitive polyclonal assays and may give lower values for glycoproteins. Some analytes have different properties, depending on the tissue or body fluid from which they originate. For example, luteinizing hormone (LH) and follicle stimulat- ing hormone (FSH) extracted from pituitaries are more like the forms of the hormones that circulate in serum than urinary preparations. The 3rd International Standard for hCG (IS 75/537) was widely used for assay standardiza- tion, but it was prepared from pregnancy urine and there- fore contained nicks (cleavages in the amino acid sequence) that may result in decreased immunological and biological potency. This standard has been replaced by successive IRP generations. However method differences still exist and hCG assays show variability in their ability to detect all forms of hCG equally (Sturgeon et al., 2009). The Canadian Society of Clinical Chemists published a useful review of diversity and its effects on standardization of assays for growth hormone, prolactin, hCG, LH, FSH and TSH (1992). They made a number of constructive recommendations including the following: G Serum or plasma concentrations of structurally defined polypeptides (e.g. ACTH, PTH, insulin, gastrin, antidiuretic hormone, glucagon and somatostatin) should be reported in molar units. G Two-site immunometric assays should be used for polypeptide hormone measurement. G A consensus should be sought regarding the epitopes to which antibodies should be directed for each of the polypeptide hormones. G Polypeptide hormone assay calibrators should be man- ufactured using recombinant DNA technology if possible. Even TSH assays still suffer from bias. In an IFCC evaluation of 16 assays, three assays still showed up to 39% bias (Thienpont et al., 2010). Determination of the molar concentration of heteroge- neous glycoproteins cannot be determined simply by dividing the weight by the molecular weight, because of the variation in molecular weight and the difficulty in removing residual water. METHOD-RELATED CAUSES OF STANDARDIZATION DIFFERENCES Differences between methods may occur even when pure, homogeneous analyte is available for the preparation of standards. Although the antibodies used in immunoassays are remarkably specific and have high affinity for their tar- get antigens, the lack of absolute specificity and adequate affinity in the presence of other substances are at the root cause of much method variation. This indicates that ultimately, it is the quality of the antibody that is most important in the development of a robust and unbiased assay. Sample and Calibrator Matrices The potential for variability in the molecular structure of the analyte between reference standard and patient sam- ple has been explained above. This, coupled with anti- body heterogeneity, can give rise to differences between immunoassays standardized using the same reference material. However such differences can also be caused by the non-analyte constituents of the standard and samples (the matrix). The influence of the matrix on the mea- surement of analyte concentrations is well known, and there is a thin line between the concepts of matrix effect, sample interference, and cross-reactivity. A matrix effect is a consistent bias in analyte determinations between two sources of matrix, such as between serum and plasma, or serum and charcoal-stripped serum. It is often used to describe a known source of bias with an unknown cause. The most important type of matrix effect is any that occurs between the matrix used to prepare the calibra- tion curve, and the matrix of the test samples. Sample interference describes an action of a known substance that can be isolated and used to cause bias if added to a previously unaffected sample. Sample interference may be the root cause of a matrix effect. Many years ago I discovered that the consistent bias of an assay in an exter- nal quality assurance (proficiency testing) scheme was caused by the presence of excessive levels of non-esterified fatty acids in the control samples, due to sample decom- position. Until the cause was identified, this was best described as a matrix effect. But in reality, it was a sample interference. As another example, many immunoassays give different analyte concentrations in paired serum and plasma samples (i.e. each blood sample is split into two aliquots and sepa- rately processed into serum and plasma). This is typical of a matrix effect. Yet the actual percentage difference may be sample or patient-specific. This is a characteristic of sample interference. Complement in samples may bind to antibodies, especially IgG2 subclass monoclonal antibod- ies, interfering with the binding to analyte. Differences in results between serum and plasma may be due to inhibi- tion of the complement activity by chelating agents in the anticoagulant in plasma collection tubes. Urine sample measurements tend to be significantly biased when serum or plasma standards are used for the calibration curve. This is a more extreme example of a matrix effect. Yet individual urine samples also vary enormously in terms of salt concentration. The third type of effect is cross-reactivity. This is dis- tinguishable from sample interference by the nature of the interfering molecule. If it binds to the analyte-binding site of the antibody because of structural similarity to the test analyte, giving rise to a false elevation of the test result, it is a cross-reactant. So it is conceivable that a matrix effect with an unknown cause may subsequently be characterized as a sample interference, when it is found to be sample- specific and transferable, and finally be traced to cross- reactivity, once the identity of the interfering substance has been identified.
  5. 5. 319CHAPTER 3.5 Standardization and Calibration From the above explanation, it follows that individual samples can vary within one type of sample matrix. Serum and plasma may be affected by the presence of rheumatoid factor, autoantibodies, human anti-mouse antibodies (HAMA) and cross-reacting drugs and metabolites. Fibrin- ogen in plasma may also interfere, and can displace pro- teins from solid phases. The chemicals used as anticoagulants for plasma collection (e.g. EDTA) may directly interfere in signal generation. Serum proteins, bilirubin and NADH can cause background fluorescence and hemolyzed samples may contain peroxidases from ruptured red blood cell membranes. Homogeneous assays are most susceptible to interferences in signal generation, because of the absence of a washing step to remove unbound interfering molecules. Other potential sources of interference are complement, phospholipids, heparin, non-esterified (free) fatty acids, and chemicals used in—or leaching from—sample collection devices. Samples and standards should always be carefully stored, to avoid further problems due to instability. Although sample stability may be experimentally verified with one immunoassay method, the presence of breakdown products may affect another. Sample interferences cause discrepant results for indi- vidual samples between methods. But susceptibility to the non-analyte constituents of the sample—whether matrix effects, sample interferences or cross-reactivity—can also result in consistent bias between two methods standard- ized against the same International Standard. This is due to differences in the nature of the non-analyte components between the standard matrix and the samples. For exam- ple, charcoal-stripped plasma, which is a convenient base matrix for preparing standards for steroids has had most small molecules completely removed and therefore differs from the population of patient samples for which the assay is intended. Potential cross-reactants are absent in the standard, but may be present in the patient samples. Less significant differences occur between serum samples and defibrinated, delipidized plasma, the usual source of a matrix for the manufacture of kit calibrators. Another example concerns the loss of dissolved carbon dioxide dur- ing freeze-drying of serum or plasma standards and controls, increasing the pH of the reconstituted matrix. Every part of an assay, and every step of the calibration process between the IS and the test samples, via secondary standards and the kit calibrator set, may be influenced by matrix effects. The binding between antibody and analyte may be affected by the local environment, for example pH, ionic strength, presence of protein, and level of hydropho- bic material. Enzyme activity during signal generation may also be affected, although most enzyme label based assays involve a wash step that removes interfering sub- stances. Each type of signal system has unique sample- based interferences, e.g. presence of natural fluorophores in samples can interfere in fluorescence measurement. The structure and integrity of the solid phase may also be affected. The influence of the sample matrix on antigen–antibody binding can be minimized by using a low ratio of sample to assay reagents in the incubation—although this reduces the sensitivity of the assay—and increasing protein and ionic concentration, and buffering capacity, through the assay reagents. Animal sera or immunoglobulins derived from the same species as the antiserum, can eliminate some sample- specific effects, such as HAMA (human anti-mouse antibody) interference. The effects of potential interferences on signal generation can be much reduced by an efficient separation system, including an effective wash step, particularly if a short soak stage is included, to allow loosely bound material to diffuse away from the solid phase into the wash solution. Homogeneous assays do not have a wash step and there- fore require meticulous attention to minimize matrix effects. To summarize, it is important to have a good under- standing of a particular immunoassay system’s inherent weaknesses, and ensure that assays are designed to com- pensate for them. Select the highest affinity and most spe- cific antibodies wherever the application allows it and take particular care over the selection of the matrix used for standards and calibrators. Attention to these aspects of assay design can significantly reduce method-related bias, and sample interferences. Buffers Buffer standards are very different from patient samples and are therefore unreliable for achieving accuracy in method standardization. There are also buffer compo- nents in the assay reagents, but as these are added to stan- dards and patient samples alike, they are unlikely to cause method bias directly. However, the nature of the buffer may have an influence on the conformation of proteins, and this in turn could affect antibody binding. So, careful choice of buffer, and optimization of pH, ionic strength and other active constituents can help to minimize method bias in standardization. Buffers may also include chemicals that release analyte from binding sites on carrier proteins (known as blocking agents). It is important that the concentration is chosen to release the analyte completely in a wide variety of samples and the standard matrix. All buffer constituents should be of a high purity, and free of potentially interfering contaminants. Antibodies The antibodies are at the heart of an immunoassay. Selec- tion of the highest affinity and most specific antibody available will reduce the influence of matrix effects consid- erably. However this strategy will increase the susceptibility of the assay to analyte heterogeneity. Thus, selection of the most suitable antibodies for a particular immunoassay involves a series of compromises, depending on a detailed understanding of the analyte and the components of the immunoassay system (sample/standard matrix, separation system and signal generation system). Where understand- ing is limited, there is no substitute for experimentation. Successful antibody production is dependent on a well- characterized immunogen. It can be difficult to obtain a purified source of analyte that is exactly like the analyte in the sample. As mentioned previously, hCG is normally derived from pregnancy urine, contains nicks between amino acids, and lacks some of the carbohydrate side- chains. This favors generation of antibodies that have a higher affinity with analyte in a standard or control, than in a patient sample, causing standardization problems.
  6. 6. 320 The Immunoassay Handbook Two-site immunometric assays are more specific. Monoclonal antibodies provide specificity but this may be at the expense of affinity. Low affinity antibodies are par- ticularly susceptible to matrix effects and sample interfer- ences. Monoclonal antibodies are also more likely to change their binding properties significantly if directly immobilized onto a solid phase, much reducing affinity. If antibodies can be selected to epitopes that are least likely to vary between different sources of analyte, the immunoassay will be less sensitive to analyte heterogene- ity. A promising area for standardization involves choosing a recombinant source of protein for the preparation of standards, then utilizing monoclonal antibodies specifi- cally chosen to bind epitopes common to the recombinant and naturally occurring protein. Labeled Analyte or Antibody In the context of standardization, the aim of labeling should be to modify the binding region of the labeled mol- ecule (analyte in a competitive assay, and antibody in an immunometric assay) as little as possible. This is a particu- lar challenge with competitive assays for small molecules. For example peroxidase-labeled triiodothyronine (T3) is 62 times larger than T3 alone. Separation Some separation systems are susceptible to matrix interfer- ences. The use of solid phases with efficient wash processes helps to minimize the effect. Problems tend to occur at low analyte concentrations in immunometric assays. Ineffi- cient washing can lead to spurious high signal levels due to sample constituents. Wash efficiency is influenced by the physical nature of the washing process, the formulation of the wash solution and the temperature of the solution dur- ing the wash. Over-washing may remove bound material. SPECIAL CONSIDERATIONS FOR ASSAY OF ANTIBODIES Although International Standards exist for some antibod- ies, the heterogeneity inherent in naturally occurring anti- body populations represents a major barrier to consistent standardization between methods. It is essential that each laboratory determines its own reference interval or cutoff based on a normal population. In the long term, standard- ization may be improved by the use of recombinant pro- teins as capture antigens, as is already happening in the field of infectious disease tests for blood donor screening. In this field there is also widespread use of accredited gray- zone samples and seroconversion sample sets. This helps to define the standardization of qualitative methods at the negative:positive interface. Calibration ANALYTE Calibration requires a source of pure or purified analyte of the highest grade available. Analytes that can be synthe- sized, such as steroids, drugs and small polypeptides, do not normally represent a problem, although they must be handled with extreme care, avoiding contamination and storing them exactly according to the manufacturer’s instructions. Particular care must be taken with hygro- scopic materials, and allowance may have to be made for water of crystallization for some chemicals. Pure analytes used for primary calibration should be stored in a desicca- tor and protected from light. Many immunoassay analytes of this type are dissolved in an organic solvent, such as ethanol. Such solutions should be diluted with an appro- priate aqueous matrix, to reduce the final concentration of the organic solvent to less than 1%. Most proteins of clinical importance exhibit some degree of heterogeneity in patient samples, although there are exceptions, such as alphafetoprotein (AFP). Several are glycoproteins, e.g. carcino-embryonic antigen (CEA), fol- licle stimulating hormone (FSH), luteinizing hormone (LH) and human chorionic gonadotropin (hCG). The het- erogeneous nature of proteins has created a need for Inter- national Standards, which represent a single source of standard material. While they may not always be homoge- neous, they at least provide a common, unitized source of analyte for all the methods available. In the field of allergy, the preparation of the allergen for use in immunoassays is fraught with technical and stan- dardization issues. Food allergen purification processes, such as heat or chemical modification, can alter the immu- nogenicity of allergens and render them unable to bind to allergen-specific IgE antibodies in patient samples. INTERNATIONAL STANDARDS To calibrate a new method for a protein, it is usually nec- essary to obtain an ampoule of an International Standard (IS) or International Reference Preparation (IRP). Preparation of primary standard solutions from ampoules requires great care and should be entrusted only to experienced personnel. Ideally, two ampoules should be reconstituted by different individuals on differ- ent occasions, with a check that the results from standard solutions made from the two ampoules are in good agree- ment. The standard solutions should be stored in aliquots at −70°C. The availability of ampoules of International Standards is strictly limited. A catalog of biological standards and ref- erence materials is available from the NIBSC. The lack of an IRP is a major cause of method differ- ences. Troponin I, which has been measured by ELISA for some 20 years, shows 2–5 fold differences between meth- ods because a material that can be used in all assay formats is difficult to obtain. SECONDARY STANDARDS In practice it is useful to prepare multiple sets of second- ary standards, from which future lots of calibrators can be assigned values. They act as an intermediate between the IS primary standard, which has a very limited availability, and new lots of calibrators for routine use. A range of 6–10 working concentration solutions should be pre- pared in bulk, covering the full range of the assay, by diluting a reliable source of the analyte in an appropriate matrix. The solutions should be subdivided into aliquots to prevent repeated thawing and re-freezing. These
  7. 7. 321CHAPTER 3.5 Standardization and Calibration should either be stored at −70°C, or freeze-dried. The matrix should be the same as the matrix intended for the final calibrators (e.g. defibrinated, delipidized human plasma). The secondary standards are then calibrated from the IS (or pure analyte) by making up a standard curve consisting of about 10 dilutions of the IS solution and determining the concentrations of the secondary standards from the IS standard curve. Great attention should be paid to calibration curve-fitting error, by checking the actual vs fitted concentrations of the IS dilu- tions. It may be necessary to hand-draw the calibration curves to minimize bias. Consistent ‘wobbles’ in the curves may indicate incorrect dilutions (immunoassay curves should only have a single point of inflection). Doubling dilutions should not be used, as cumulative errors may cause bias at lower concentrations. At least 20 assays should be run to obtain mean values for the sec- ondary standards. Different reagent lots and instruments should be used if possible, in an ANOVA (Analysis of Variance) experimental design. Any outlier data should be rejected. Any significant variability between reagent lots or analyzers, detected in an ANOVA analysis, should be investigated. CALIBRATORS Immunoassays require the use of calibrators in order to assign values or concentrations to unknown samples. In a classical immunoassay, a set of about six calibrators is run prior to the unknown samples, a calibration curve of signal vs concentration is plotted, and the concentrations of the unknown samples determined by interpolation (see CALI- BRATION CURVE-FITTING). This is usually carried out using a computer. The calibrator sets are made in bulk, and the values may be assigned by reference to the secondary stan- dards (in about 20 assays). Many immunoassay reagent kits contain fixed-value calibrators, which always have the same values assigned, regardless of the lot. This requires careful process control against the secondary standards during manufacture. Calibrators should ideally be prepared by using a base matrix identical to that in the test samples. A preferred material for many clinical tests is defibrinated, delipidized plasma, from a pool of donated human blood. Each donated blood sample must be separately tested for HIV and HCV antibodies, and HBsAg. One of the difficulties facing manufacturers is that a range of calibrators must be made, including one with an analyte concentration of zero. Many analytes are present in normal human serum, and preparation of a zero cali- brator requires the use of a different matrix. Possible alternatives include animal serum or a buffer solution containing protein, but both of these may give rise to matrix effects. Horse serum is particularly variable and should be avoided. Possibly the best option is to use human serum stripped of analyte by affinity chromatogra- phy, using antibody immobilized onto a column. How- ever trace amounts of antibody may leach from the column. Some low molecular weight analytes may be removed from serum by stripping using charcoal or ion- exchange resin, but this removes other small molecules, changing the matrix considerably. STANDARD AND CALIBRATOR MATRICES The matrix of a calibrator needs to behave in a similar way to the sample matrix, keeping less soluble analytes in solu- tion or bound to a carrier protein, and providing a back- ground level of proteins that may play a role in the incubation or separation stages of the assay. There are two conflicting requirements for a calibrator matrix: 1. The matrix should be consistent from lot to lot. 2. The matrix should reflect any non-analyte constitu- ents of patient samples that have a background effect in the assay. The ideal way to achieve (1) would be to use a buffer containing, for example, 1% bovine serum albumin, but this is often inadequate in meeting the second require- ment. Animal serum is occasionally used, but can suffer from lot–lot variability. For clinical applications, human serum (or defibrinated, delipidized plasma) is the preferred base matrix. The effects of the natural variation between donors are best minimized by using pooled collections from a number of individuals. REDUCED AND STORED CALIBRATION CURVES Unless many samples are being run at a time, the need to run a set of six calibrators in duplicate, to derive a calibra- tion curve, is wasteful of reagents. In small assay runs, or on random-access analyzers, more reagents could be used up for the calibrators than for the test samples. For this reason, a variety of reduced calibration curve methods have been developed. Some immunometric assays with lin- ear calibration curves require only one or two calibrators to be run. The linearity is achieved by immobilizing high levels of capture antibody on the solid phase, however this approach only works for some assays. A major step for- ward: stored calibration curves, came with the introduc- tion of stable, fully automated analyzers with stable reagents that only required calibration at periodic inter- vals. For example six calibrators are run in duplicate and the calibration curve is computed. The curve is stored in the analyzer’s memory and used to determine the concen- tration of any samples tested using the same lot of reagents. Typically these stored calibration curves are stable for at least 14 days, but some systems have calibration stabilities of several months. The next step was to extend the life of the full calibra- tion with a reduced set of one or two calibrators, used to correct the main calibration curve, e.g. for long-term ana- lyzer fluctuations. A good example is the Abbott IMx™ single point ‘MODE 1’ correction. The ACS™:180 took this a stage further, with the introduction of encoded master calibration curves. They are derived from a series of assays carried out at the man- ufacturer’s QC laboratory on each new lot, and encoded in a barcode that is read by a barcode reader on the ana- lyzer. Periodic recalibration is accomplished by running two multianalyte calibrators (or adjusters). The computer uses the signal levels for the two calibrators to adjust the master curve, compensating for analyzer–analyzer differences and changes in the kit reagents over the shelf-life.
  8. 8. 322 The Immunoassay Handbook Encoded master calibration curves are now used on many random-access analyzers. The master calibration data, and other information, is transferred from the manufacturer to the analyzer using conventional barcodes, two-dimensional barcodes (which can encode more data and are more robust), magnetic cards, smart cards or floppy disks. Normally the adjusting calibrator values are assigned by the manufacturer using several lots of reagents, then used in the clinical laboratory with later lots. However, small variations between reagent lots can cause inconsistencies in calibrator value assignment. The Vitros ECi™ system has lot-specific calibrator values, linking the value assign- ment of the calibrators with the determination of the mas- ter curve for each lot. These values are encoded with the master curve data on a magnetic card. To achieve stable calibration curves the analyzer must maintain consistency and stability in incubation temperature (including warm-up rate), signal measurement and other variables that can affect signal levels. It must also withstand changes in ambient laboratory temperature and humidity within specified limits. Kits within a lot must give consistent results even if they have been shipped at different times and thus subjected to different conditions while in transit. To ensure consistency, some manufacturers have introduced cooled shipment for distribution. Any generic reagents (e.g. substrate or wash reagent) must also give constant signal lev- els across multiple lots and throughout their shelf-lives. Many immunoassays for drugs of abuse and infectious diseases are run using a single calibrator or ‘control’. This is run to determine a cutoff level between ‘negative’ and ‘positive’. Normally, positive and negative controls are also run as unknowns to check the assay calibration. Tests for use in doctors’ offices, pharmacies and in the home are self-calibrating, typically generating color in the presence of a clinically significant concentration of analyte. RECOVERY AND DILUTION Once a method has initially been calibrated, recovery effi- ciency and dilution characteristics should be checked. These are the key indicators of calibration quality. These and other checks are described in METHOD EVALUATION. References and Further Reading Apple, F.S. Clinical and analytical standardization issues confronting cardiac troponin I. Clin. Chem. 45, 18–20 (1999). Canadian Society of Clinical Chemists Position paper: standardization of selected polypeptide hormone measurements. Clin. Biochem. 25, 415–424 (1992). Dikkeschei, L.D., de Ruyter-Buitenhuis, A.W., Nagel, G.T., Schade, J.H., Wolthers, B.G., Kraan, G.P.B. and van der Slik, W. GC–MS as a reference method in immunochemical steroid hormone analyses. J. Clin. Immunassay 14, 37–43 (1991). Gosling, J.P., Middle, J., Siekmann, L. and Read, G. Standardization of hapten immu- noprocedures: total cortisol. Scand. J. Clin. Lab. Invest. 53 (Suppl 216), 3–41 (1993). Hilgers, J., von Mensdorff-Pouilly, S., Verstraaten, A. et al. Quantitation of poly- morphic epithelial mucin: a challenge for biochemists and immunologists. Scand. J. Clin. Lab. Invest. 55 (Suppl 221), 81–86 (1995). Jeffcoate, S.L. Analytical and clinical significance of peptide hormone heterogeneity with particular reference to growth hormone and luteinizing hormone in serum. Clin. Endocrinol. 38, 113–121 (1993). Kallner, A., Magid, E. and Albert, W. (eds), Improvement of Comparability and Compatibility of Laboratory Assay Results in Life Sciences, 3rd Bergmeyer Conference: Immunoassay Standardization, Scand. J. Clin. Lab. Invest. 51, Suppl, vol. 205, (1991). Kallner, A., Magid, E., Ritchie, R. (eds), Improvement of Comparability and Compatibility of Laboratory Assay Results in Life Sciences, 4th Bergmeyer Conference: Proposals for Two Immunomethod Reference Systems: Cortisol and Human Chorionic Gonadotropin, Scand. J. Clin. Lab. Invest. 53, Suppl, vol. 216 , (1993). Marcovina, S.M., Albers, J.J., Henderson, L.O. et al. International federation of clinical chemistry standardisation project for measurements of A-1 and B. III. Comparability of apolipoprotein A-1 values by use of international reference material. Clin. Chem. 39, 773–781 (1993). Mire-Sluis, A.R., Das, R.G. and Padilla, A. WHO cytokine standardization: facilitating the development of cytokines in research, diagnosis and as therapeutic agents. J. Immunol. Methods 216, 103–116 (1998). NCCLS. National Reference System for the Clinical Laboratory. Reference system: clinical laboratory. NRSCL13-P (NCCLS, Wayne, Pennsylvania). NCCLS. National Reference System for the Clinical Laboratory. Terminology and definitions for use in NCCLS documents. NRSCL8-A (NCCLS, Wayne, Pennsylvania). NCCLS. A candidate reference method for serum digoxin. I/LA9-T (NCCLS, Wayne, Pennsylvania). NCCLS. National Reference System for the Clinical Laboratory. Reference meth- ods, materials and related information for the clinical laboratory. NRSCL12-P (NCCLS, Wayne, Pennsylvania). NIBSC. Catalogue of Biological Standards and Reference Materials (National Institute for Biological Standards and Control, Potters Bar, UK, www.nibsc.ac.uk). Panteghini, M. Recent approaches in standardization of cardiac markers. Clin. Chim. Acta 311, 19–25 (2001). Seth, J. Standardization of protein hormone immunoassays. Ann. Clin. Biochem. 33, 482–485 (1996). Stamey, T.A. 2nd Stanford conference on international standardization of prostate-specific antigen immunoassays, 1994. Urology 45, 173–184 (1995). Stenman, U.-H., Bidart, J.-M., Birken, S., Mann, K., Nisula, B. and O’Connor, J. Standardization of protein immunoprocedures: choriogonadotropin. Scand. J. Clin. Lab. Invest. 53 (Suppl 216), 42–78 (1993). Stenman, U.-H. Standardisation of immunoassays. In: Principles and Practice of Immunoassay, (eds Price, C.P., and Newman, D.J.) pp. 243–268 (Macmillan, London, 1997). Storring, P.L. Assaying glycoprotein hormones—the influence of glycosylation on immunoreactivity. Trends Biotechnol. 10, 427–432 (1992). Sturgeon, C.M. and McAllister, E.J. Analysis of hCG: clinical applications and assay requirements. Ann. Clin. Biochem. 35, 460–491 (1998). Sturgeon, C.M., Berger, P., Bidart, J.-M. et al. Differences in recognition of the 1st WHO international reference reagents for hCG-related isoforms by diagnostic immunoassays for human chorionic gonadotropin. Clin. Chem. 55, 1484–1491 (2009). Tate, J.R., Bunk, J.M., Christenson, R.H. et al. Standardisation of cardiac troponin I measurement – past and present. Pathology 42, 402–408 (2010). Taylor, S.L., Nordlee, J.A., Niemann, L.M. and Lambrecht, D.M. Allergen immunoassays – consideration for use of naturally occurring standards. Anal. Biochem. Chem. 395, 83–92 (2009). Thienpont, L.M. and Van Houcke, S.K. Traceability to a common standard for protein measurements by immunoassay for in vitro diagnostic purposes. Clin. Chim. Acta 14, 2058–2061 (2010). Thienpont, L., Siekmann, L., Lawson, A., Colinet, E. and De Leenheer, A. Development, validation and certification by isotope dilution gas chromato- graphy-mass spectrometry of lyophilized human serum reference materials for cortisol (CRM 192 and 193) and progesterone (CRM 347 and 348). Clin. Chem. 37, 540–546 (1991). Thienpont, L.M., Van Uytfanghe, K., Beastall, G. et al. Report of the IFCC work- inggroupforstandardizationofthyroidfunctiontests;part1:thyroid-stimulating hormone. Clin. Chem. 56, 902–911 (2010). Vihko, P. and Wagener, C. Structure and genetic engineering of antigens and antibodies: applications in immunoassays. Ann. Biol. Clin. 50, 607–611 (1992). Whicher, J.T., Ritchie, R.F., Johnson, A.M. et al. New international reference preparation for proteins in human serum (RPPHS). Clin. Chem. 40, 934–938 (1994). WHO and Van Aken, W.G. WHO Expert Committee on Biological Standardization: 56th Report, (2007). (Available for download from WHO website). Wood, W.G. ‘Matrix effects’ in immunoassays. Scand. J. Clin. Lab. Invest. 51 (Suppl 205), 105–112 (1991). REFERENCES CONTAINING DISCUSSIONS ABOUT STANDARDIZATION Ellington, A.A., Kullo, I.J., Bailey, K.R. and Klee, G.G. Antibody-based protein multiplex platforms: technical and operational challenges, Clin. Chem. 56, 186–193 (2010). Good section on standardization and QC of multiplex assays. Gorovitz, B. Antidrug antibody assay validation: industry survey results. AAPS J. 11, 133–138 (2009). Resch-Genger, U. Standardization and Quality Assurance in Fluorescence Measurements II: Bioanalytical and Biomedical Applications (Springer Series on Fluorescence, vol. 2). (Springer, 2008). Includes a section on fluorescence immunoassay microarray standardization. Sztefko, K. Immunodiagnostics and Patient Safety (Walter de Gruyter & Co, Berlin 2011). Good general section on standardization as a review.

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