Quality management in IgE-based allergy diagnostics
-
Jörg Kleine-Tebbe
,
Lars K. Poulsen
Abstract:
Assays for total and allergen-specific (s) IgE are essential serological tests in the diagnostic work-up of immediate type hypersensitivity reactions and atopic diseases. Technical performance characteristics and clinical utility of IgE tests have been published in international guidelines. In the USA and in Europe, IgE tests are mainly performed by accredited medical laboratories and in Germany they are also performed by allergists carrying an OIII-limited license. Both have to perform continuously internal and external quality control measures including proficiency trials twice a year (in Germany). Due to the heterogeneity of the assay’s core allergen reagents, complex extracts and more recently defined allergenic molecules, and heterologous assay calibration, the results of qualitative and quantitative sIgE tests from different diagnostic manufacturers can vary considerably. Proficiency trial results are subsequently grouped according to each assay type. Passing acceptance criteria depend on national rules and regarding quality management. Future challenges include a more valid quantification of sIgE which would allow true comparisons with the international units for total IgE, and the use of harmonized allergen reagents for the most important allergen sources, which have hampered inter-assay comparability in the past.
Zusammenfassung:
Methoden zur Bestimmung des Gesamt- und spezifischen Serum-IgE sind wichtige serologische Tests zur Abklärung allergischer Soforttyp-Reaktionen und atopischer Erkrankungen. Testeigenschaften und klinische Tauglichkeit von IgE-Bestimmungsmethoden wurden in internationalen Leitlinien abgebildet. In den USA und in Euroapa werden IgE-Tests überwiegend durch akkreditierte medizinische Labors und in Deutschland auch durch Allergologen mit einer OIII-Zulassung durchgeführt. Beide haben regelmäßige interne und externe Qualitätsmaßnahmen durchzuführen. Dazu gehört in Deutschland zweimal pro Jahr die Teilnahme an Ringversuchen. Durch die Heterogenität der zentralen Reagentien, den Testallergenen - komplexe Extrakte oder zunehmend definierte Allergenmoleküle -, und heterologe Testkalibration können die Ergebnisse von qualitativen und quantitativen sIgE-Tests verschiedener Diagnostikahersteller erheblich voneinander abweichen. Die Ergebnisse von Ringversuchen werden daher gruppiert je nach Testmethode ausgewertet. Bestehensregeln sind von nationalen Vorschriften zum Qualitätsmanagment abhängig. Zukünftige Herausforderungen betreffen eine allgemein gültige sIgE-Quantifizierung, die einen echten Vergleich mit den internationalen Einheiten für das Gesamt-IgE gestattet, und die Verwendung harmonisierter Allergenreagentien für die wichtigsten Allergenquellen, die in der Vergangenheit die Vergleichbarkeit zwischen den Tests erschwert haben.
Reviewed Publication:
Sack U. Conrad K.
From RAST to modern immunoassays for allergen-specific IgE
Soon after its identification in the late 1960s, [1–3] allergen-specific immunoglobulin E (IgE) antibodies were detected by a noncompetitive, heterogeneous, immunoradiometric assay that used allergen extracts covalently linked to microcellulose particles (allergosorbent) [4]. The assay was referred to as the Radio-Allergo-Sorbent-Test (RAST) and it has subsequently evolved into various assay formats, some which have allowed automation of allergen-specific IgE measurements from serum and other human specimens. Two lines of product development have emerged:
Replacement of radioisotope-labeling of the anti-IgE antibody by other physico-chemical detection principles: enzyme-catalyzed development of absorbance (EAST) or fluorescence (FEIA), chemiluminescence or time-resolved fluorescence (dissociation-enhanced lanthanide fluoroimmunoassay – DELFIA).
The principles and current technical and quality control issues associated with modern total and allergen-specific IgE assays have been thoroughly examined in a 2016 international laboratory consensus document [8]. Clinical guidelines (http://www.dgaki.de/leitlinien/aktuelle-leitlinien/) and overviews [9] provide additional recommendations for the user as to when to utilize and how to interpret IgE tests in the diagnostic work-up of IgE-mediated immediate-type hypersensitivity reactions to aeroallergens, drugs, foods and insect venoms. Individual guidelines address specific atopic diseases that include IgE-mediated food allergic reactions [10] and atopic eczema [11].
Biophysical separation of individual allergens in an extract in 1- or 2-D patterns, such as crossed immunoelectrophoresis [12], or by molecular weight and/or isoelectric point [13] allow the resulting pattern to be used as allergosorbent, resulting in a semiquantitative assay that allows the measurement of IgE-binding to different individual allergens in the mixture. Recently, the introduction of purified native or recombinant allergen molecules for use IgE diagnostics has opened the new field of “Molecular Allergology” [14]. Enhancing the assay format from a singleplex [9] to a multiplex format [15] has permitted the establishment of new technologies that have the potential to permit targeted allergen-specific therapies in the future [16]. This is not to be confused with the singleplex/multiplex assay which describes the spectrum of allergen reagents that are available today. This spectrum spans from single allergen components, to multi-component extracts of a single biological species to panels of different allergen extracts or components mixed together and immobilized on the same solid-phase.
The following paragraphs focus on current concepts of quality control of serological total and allergen-specific IgE assays. Practical hints for effective quality control and organizations that offer proficiency testing in Germany (mandatory twice a year), Europe and the USA for insuring a robust quality control program are overviewed. Finally, unresolved issues of variable allergen-specific IgE results that are obtained from different assay formats and manufacturers due to calibration method differences and reagent heterogeneity will be discussed.
Regulatory framework
Germany (Table 1): In general, laboratory testing and quality control issues are guided by the revision of the “Guideline of the German Medical Association on Quality Assurance in Medical Laboratory Examinations” (unauthorized English translation in [17]). It was officially published in the German language as an updated guidance document “Richtlinie der Bundesärztekammer zur Qualitätssicherung laboratoriumsmedizinischer Untersuchungen” (Rili-BÄK) [18]. Laboratories performing total serum IgE and qualitative as well as quantitative allergen-specific IgE assays have to perform proficiency trials twice a year, as outlined in table B 2-2 of the Rili-BÄK. Larger laboratories are usually accredited according to the DIN EN ISO 15289 standard, requiring proper quality management and regular certificates of successfully passed proficiency trials.
Institutions, legal requirements and information sources.
| Region | Regulatory body | Requirements | Sources-standardsa |
|---|---|---|---|
| Germany | German Medical Association (“Bundesärztekammer”) | Internal quality control (calibration control, assay control) External quality control (Proficiency testing) | RiLiBÄK guideline http://www.bundesaerztekammer.de/fileadmin/user_upload/downloads/Rili-BAEK-Laboratoriumsmedizin.pdf |
| Europe | Country specific competent authorities | Varying due to national legislation and ordinances | Not harmonized |
| USA | US centers for medicare and medicaid services Federal center for standards and quality | – Personnel qualifications (training-experience) – Laboratory operation – Assay calibration, quality control, proficiency testing – Test system troubleshooting – Equipment maintenance – Interpretation-reporting – Personal health information management | Clinical Laboratory Improvement Amendments of 1988 (section 353, PublicHealth Service Act (42 U.S.C. 263a) Clinical Laboratory Standards Institute ILA/20) (method guidance document) |
aStandards that apply to clinical laboratory testing performed on specimens from humans.
In Germany, serologic allergy diagnostics are not only part of the (routine) work-up in regular medical laboratories, but also considered integral part of the subspecialty of allergy (official German term: “Zusatzbezeichnung Allergologie”). Assays for total and allergen-specific IgE provide essential services that shape this subspeciality, particularly regarding proper indication, performance and interpretation of these tests. Serologic IgE assays are therefore not only a vital part of the current and future education policy (“Weiterbildungsordnung”) for allergists, but they are also reflected in the current mandate from the public (“Versorgungsauftrag”). Subsequently, they belong to the core competence of the discipline and can be run by specialized and qualified allergists who possess an OIII-limited laboratory approval. Noteworthy, they have to fulfill the Rili-BÄK requirements including two proficiency trials per year (further details see below).
Europe (Table 1): The European Union Directive 98/79/EC [19] regulates the product quality, and establishes the CE mark as a necessary requirement for marketing in vitro diagnostic reagents or kits on the European market. A number of standards have been established to specify various aspects of product quality of in vitro tests.
However, when commercial assays are used, there are no uniform European standards governing the performance of clinical laboratories. Instead this is regulated by the individual European states.
Two types of quality assurance are available to laboratories:
certification (where a third party gives written assurance that a product, process or service conforms to specific requirements) and
accreditation (where an authoritative body gives formal recognition that a body or a person is competent to carry out a specific task).
Whereas the former sets requirements for a quality management system (QMS) such as ISO 9001, the latter sets requirements for a QMS and requirements regarding technical and analytical competence. Typical standards for the latter are ISO 17025 and ISO 15189. There are multiple certification bodies in each country, but just a single accreditation body, and in many European countries there is a steady increase in clinical laboratories being accredited according to the ISO 15289 standard [20]. No data exist on how many laboratories that are performing IgE-analyses are accredited, but several investigations from France, where accreditation will become compulsory in 2016 [21] illustrate the process involved in the application of ISO 15189 for the analysis of specific IgE antibodies [22].
USA (Table 1): Clinical laboratories in the USA are governed by the federal regulatory standards detailed in the Clinical Laboratory Improvement Amendments of 1988 (CLIA-88) [23]. CLIA-88 licensure insures that laboratory provides accurate, reliable and timely test results. In terms of quality assurance, CLIA-88 mandates that all diagnostic allergy laboratories have a rigorous in-house quality control program and they successfully participate in an external proficiency survey that minimally involves the testing of up to six serum specimens three times a year (every 17 weeks) for total IgE and IgE antibodies specific for five allergen sources. Failure to generate IgE data that is within the mean±2SD of peer laboratories performing the same assay can result in laboratory license removal. The most extensive diagnostic allergy proficiency program in North America is conducted by the College of American Pathologists [24].
Quantitative total IgE in international units
The quantitation of total serum IgE involves the binding and detection of IgE molecules with a pair of IgE specific antibodies in an immunometric (sandwich) assay format (Figure 1A). These assays are all calibrated to the World Health Organization (WHO) International Reference [25] Preparation for human IgE which has recently a transition from WHO 75/502 to WHO 11/234. Various detection systems are employed for total IgE measurements, based on – photometric (i.e. Hytec®, Hycor; total IgE, BDL), -fluorometric (i.e. ImmunoCAP®, ThermoFisher), -chemiluminescent (i.e. Immulite®, Siemens; Optigen®, Hitachi), and immunoturbidimetric (i.e. Response®, DiaSys) endpoints.
Principle IgE assay formats.
(A) General total IgE assay format. (B) “Classical” solid phase assay format for the detection of sIgE. (C) Fluid phase assay format for sIgE. (D) Reverse phase assay format for sIgE. Explanations see text.
As a result of differences in assay design, calibration, the source and affinity of anti-IgE reagents and various detection endpoints, total IgE levels might be expected to vary among laboratories. However, use of the same IRP WHO 11/234 minimizes significant systematic bias and allows remarkable agreement in total serum IgE results among the majority of quantitative assays. Historically total serum IgE measurements were performed because of their clinical association with the presence of asthma [26]. However, the overlap in total serum IgE levels among non-atopic and atopic individuals (Figure 2) diminished their use for a period. Currently total serum IgE measurements are resurging due to two principal factors. First, their use is critical in the proper dosing of patients receiving anti-IgE therapy with omalizumab [27]. Second there is increased appreciation that at the percentage of total IgE that is specific for a given allergen is one of the four humoral immune response parameters of IgE in addition to IgE antibody affinity, concentration and epitope specificity that impacts on effectiveness in eliciting clinically evident allergic symptoms [28]. The ratio of specific to total IgE provides an indicator of “immuno-dominant” allergen sources and allows one to rank the proportion of allergen-specific activities within individuals [29].
Result reporting of total IgE levels.
Total IgE levels are expressed as IE/mL (equivalent to kU/L, 1 kU/L=2.4 ng/mL IgE), using the WHO IRP for total serum IgE. White curved area: population of sera from non-atopic individuals with rather low, but variable total serum IgE levels. Light gray area: population of sera from atopic subjects with moderately high, but also highly variable total IgE levels. Note: considerable overlap (“grayzone”) and poor discrimination between non-atopic and atopic individuals with either cut-off for total IgE, i.e. 25 or 100 kU/L. Dark gray area: population of sera containing widely distributed high total IgE levels, quite common in atopic dermatitis (AD) or ABPA. ABPA, allergic bronchopulmonary aspergillosis; IE/mL, International Units per mL; IgE, immunoglobulin E.
Assay formats for allergen-specific IgE antibody detection
Various assay formats (design principles in Figure 1B–D) [30] are internationally marketed for the detection of allergen-specific IgE. While the US market offers limited assay formats due to the required clearance by the FDA, the German and European markets show a sometimes confusing and quickly changing patchwork of available methods for the detection of allergen-specific IgE with vast regional differences.
Design principles of allergen-specific IgE assays
“Classical” solid phase allergen-specific IgE immunoassays (Figure 1B) are designed to bind all antigen-specific antibodies, and non-allergen binding components are then removed with buffer washes. Specific IgE antibody is detected by labeled anti-IgE antibodies. With the limited amount of antigen that can be immobilized on certain solid phase matrices (e.g. chip), this assay format will be affected by competitive inhibition by allergen-specific IgG that occurs in high titers following allergen-specific immunotherapy. With molar excess of antigen, this assay format is less prone to IgG-antibody inhibition.
Fluid phase allergen-specific IgE immunoassays (Figure 1C) can offer quick antibody binding kinetics but their robustness to interference from non-IgE specific antibodies also depend on the amount of applied allergen reagent.
Reverse type allergen-specific IgE immunoassays initially bind the entire IgE repertoire with immobilized anti-IgE Fc (Figure 1D). Following a buffer wash, bound allergen-specific IgE is then detected with a labeled allergen reagent. The assay is not adversely impacted by IgG-inhibition, but it has limitations in the case of very high total serum IgE levels that can exceed the anti-IgE binding capacity and thus produce potentially falsely low results.
Allergen reagents: essential assay variables that account for discrepant results
Regardless of which of the assay formats that is used, the most important component is the allergenic reagent, either a solid-phase allergosorbent or liquid-phase labeled allergen. Until recently, allergenic extracts from a plethora of allergen sources were principally used for allergen-specific IgE detection. Extracts of inhalant allergens (tree, grass and weed pollen, mites, mammals, molds), – food stuffs (various plant and animal derived foods), Hymenoptera venoms (bee and wasp species), natural rubber latex and other sources are complex mixtures of allergenic molecules (often proteins in minute to high amounts) and non-allergenic material.
Due to different source materials and extraction methods, extracts vary in their overall allergenic potency as well as their allergen composition (number and amount of extracted allergens). Ultimately, the allergen reagent, based on particular in-house procedures, accounts for most of the discrepancies between allergen-specific IgE assays from different diagnostic manufacturers. As a consequence, binding of an individual’s IgE repertoire will vary considerably and lead to non-identical quantitative IgE antibody results. This variability in allergen-specific IgE results that are obtained from different IgE assay formats despite the use of “identical” allergen (sources) is presumably the most important unresolved issue in achieving harmonization among serological quantitative in-vitro allergy diagnostics (see below).
Qualitative and quantitative result reporting of IgE antibody levels
Serological, log-distributed allergen-specific IgE results (Figure 3) are, depending on the assay design and features (see Table 2), reported either as qualitative (absence or presence), semi-quantitative (“classes”) or quantitative results. The arbitrarily assigned “classes” were introduced with the original RAST to indicate relative semi-quantitative levels and broadly categorize allergen-specific IgE concentrations. They roughly paralleled the semi-quantitative scheme used in evaluation of skin prick tests in the past; the latter one are now preferably documented by providing the average diameter (mm) of the wheal. Use of quantitative IgE antibody levels in kUA/L (Figure 1) that are based on interpolation from a total IgE reference curve should be encouraged [31]. An international IgE reference preparation which is calibrated in units that are traceable to mass measures (2.4 ng/mL IgE≙1 IU/mL=kU/L) is available for use in total serum IgE assays [25].
Result reporting of allergen-specific IgE levels.
(A) Quantitative: allergen-specific IgE levels expressed as units of specific IgE, kUA/L (A stands for allergen-specific), using the WHO IRP for total serum IgE determination involving heterologous interpolation as a calibration scheme. (B) Semi-quantitative: due to the German Medical Association guideline, Richtlinie der Bundesärztekammer (RiliBÄK), this term is no longer permitted; specific IgE levels given only in classes are considered as qualitative evaluations. (C) Qualitative (positive or negative). White curved area: population of serum samples with no allergen-specific IgE (levels below the quantitation limit of 0.1 kUA/L). Dark gray area: population of positive serum samples with logarithmic distribution of allergen-specific IgE levels above the detection limit of 0.1 kUA/L. IgE, Immunoglobulin E; IRP, International Reference Preparation; WHO, World Health Organization.
General classification scheme for allergen-specific IgE assays (with permission from Hamilton et al., CLSI document I/LA20-A3, 2016) [8].
| Classification | Result presentation | Standardization method | References calibrators and controls |
|---|---|---|---|
| Qualitative | – Reactive or nonreactive – Positive or negative An “indeterminate” zone may be included | – Single or dual calibrators to normalize assay | – One reference sample – Negative and positive control |
| Semi-quantitative | – Arbitrary units or Classes | – Single or dual calibrators to normalize assay | – Negative and bilevel positive controls (occasionally tri-level controls) – References unique for the test system made from pooled patient sera |
| Quantitative | – Units related to a generally recognized IRP (e.g. WHO IgE IRP) | – Multipoint standard curve used for interpolation | – Tri-level positive controls – References calibrated towards a qualified reference preparation |
IRP, international reference preparation.
Heterologous calibration providing allergen-specific IgE mass units
Presently, there are no internationally recognized allergen-specific IgE reference preparations. Thus, in modern quantitative IgE antibody assays, a total IgE calibration curve is used to convert the assay’s measured response into quantitative allergen-specific IgE antibody units (Figure 3): kUA/L (where “A” stands for “allergen-specific”, thereby distinguishing its units from the internationally standardized kU/L=IU/mL for total IgE determination). The measurement signals obtained for allergen-specific IgE concentrations are thus always interpolated from a total IgE reference curve by “heterologous” interpolation (details on calibration schemes in Table 3).
Different assay calibration schemes and consequences for singleplex and multiplex allergen-specific IgE-assays (with permission from Hamilton RG and Kleine-Tebbe J, Methods for IgE Antibody Testing: Singleplex and Multiplex Assays. In: EAACI Molecular Allergology User’s Guide. Ped Allergy Immunol 2016, in press).
| Heterologous calibration | Allergen-specific calibration | |
|---|---|---|
| Standard curve | IgE values traceable to a total serum IgE reference preparation | Allergen-specific curve for each allergen specificity |
| International reference preparation | WHO International Standard Immunoglobulin E (IgE), human serum NIBSC code: 11/234 [11] | Not available for allergen-specific human IgE |
| Potentials (pro) | Interpolation of allergen-specific IgE to any allergen specificity, as long as total IgE and allergen-specific IgE antibody dilute out in parallel (similar binding characteristics assumed) | Theoretically ideal individual standard curve considering potential differences in IgE binding to various allergenic reagents |
| Limitations (con) | Slight error (<10%) due to minor differences in the binding characteristics of IgE to the anti-IgE reagent and the allergen reagent | Standard curve for every allergen specificity needed (impossible task) |
| Examples | Thermofisher ImmunoCAP, Siemens Immulite, Fooke Allerg-O-Liq, Hycor.Hytech 288 | Abbott Matrix (stopped in 1992) |
| Potential use in multiplex assay formats | Difficult in micronized assays with minute amount of allergen (and anti-IgE) reagents Alternative heterologous calibration scheme for microarrays: Single serum calibrator that has defined IgE antibody levels for 10 different aeroallergen spots | Not feasible |
| References | Hamilton et al. [8] (CLSI consensus) |
Heterogeneity of results from different IgE assay platforms
As a consequence of using different anti-IgE reagents and in-house calibration schemes, the different IgE assays platforms perform slightly different. This may account for <10% of the variation observed between assays representing a source of error that needs to be accepted as a result of adopting heterologous calibration as a calibration strategy (Table 3). However, the principal reason for heterogeneous results is believed to stem from differences between various allergen reagents (even in case of the same allergen source) that are used by different assay developers. This has direct implications for (particularly external) quality control measures since proficiency test results from different laboratories that use the identical assay system from the same manufacturer need to be grouped for peer group comparison.
Quality assurance and quality control: general principles
Quality assurance (QA) is process oriented and is equivalent to process management. In contrast, quality control (QC) is product oriented and might be best described as a product test. Testing for quality does not assure quality, but controls for it.
The validity of results from total serum IgE and allergen-specific IgE antibody assays should be based on three areas of quality assurance and control (I/LA20-A3):
QA and QC during reagent and assay development and production by the manufacturer.
Internal QC by the clinical laboratory user to demonstrate that each assay is “in control”.
External QC involves regular participation in an external quality assurance or proficiency survey.
Manufacturer’s QA and further QC (A) can minimize the need for subsequent internal intra-laboratory QC testing (B) in the clinical laboratory. Internal QC (B) can help to improve results in subsequent external QC, i.e. laboratory proficiency programs (C).
Quality management in manufacturing
The assay’s complexity and degree of quantitation shape the developer’s program for QA and QC during development and manufacturing.
After clearance of the individual reagents, the assembled assay should be tested with sera from clinically non-allergic (IgE antibody-negative) and allergic (IgE antibody-positive) individuals (see I/LA20-A3) [27]:
For qualitative IgE assays, testing of a panel of allergen-specific IgE antibody positive and negative control sera should generate results that identify the correct IgE antibody assignment (positive or negative). The goal of these quality assurance analyses is to document the appropriateness of the positive threshold and validate the allergen specificity of the assay.
Additionally, for semi-quantitative and quantitative IgE antibody assays, the calibration curve should be validated with at least three quality control sera containing levels of IgE antibody that cover a) two to three times the detection limit, b) the assay range midpoint, and c) within 10% of the upper extreme plateau level.
For quantitative IgE assays only, reproducibility of the test system should be documented by the manufacturer with
precision profiles (intra-assay and inter-assay coefficients of variation (IACV, IECV) in relation to the strength of the signal),
verification of the assay’s limit of detection (LoD) and quantitation (LoQ), and
linearity using a dilution-recovery analysis when each new lot of reagent is produced, prior to release to the user.
After development and introduction of a new IgE assay format, its features (1–3) should be scientifically published, which is rarely the case, or at least be accessible so the assay’s performance characteristics can be reviewed by interested users, i.e. customers, physicians as well as regulators. Ultimately, the scientific community or the routine clinical laboratory does not need to repeat the extensive validation and quality assurance testing performed by the manufacturer to successfully employ the commercially available reagents in their IgE antibody autoanalyzer.
Technical and clinical evaluation: gap between expectations and reality
Manufacturers should not only perform a thorough technical evaluation of their particular allergen-specific IgE assay format, but provide the outcome in published manuscripts or another accessible form. Scientific journals seem to neglect the value of purely technical evaluations and should therefore consider such results more seriously for potential publication.
Clinical evaluations of allergen-specific IgE assays can practically be obtained only for a selected number of clinically important allergen specificities. Diagnostic sensitivity and specificity, as well as positive and negative predictive values (PPV and NPV) have been addressed for certain allergen sources. However, allergen-specific assays can only determine allergic “sensitization” (risk of allergy), being clinically relevant only in case of corresponding symptoms. Thus, negative results of technically well performing assays are helpful to rule out a particular sensitization (and subsequent allergy), but positive results have to be carefully interpreted by the physician, knowing the patient’s history, for their subsequent clinical relevance.
Internal quality management of IgE assays
General goals
Internal quality programs are useful and required in the US and globally in all accredited laboratories. Individual strategies can vary depending on the assay format and equipment used. General guidance on developing an internal quality control program and specific strategies for the internal QC of total serum IgE and allergen-specific IgE antibody assays are presented in the I/LA20-A3 guidance document [27] and summarized in the next paragraph.
Suggestions, samples and advices
At a minimum, a single IgE antibody positive control serum should be analyzed in each assay. Some laboratories analyze a group of 2–15 common allergen specificities (i.e. European aero allergens like birch pollen, timothy grass pollen, cat dander, Dermatophagoides pteronyssinus or US allergens like common ragweed, oak tree, Alternaria tenuis, dog dander, and Dermatophagoides farinae). A serum pool is then prepared containing IgE antibody to all 2–15 specificities. It is analyzed against all 2–15 allergen-containing reagents separated throughout each assay run. Alternatively, manufacturer-produced controls can be run in each assay. This approach provides confirmation of the calibration portion of the assay, and it validates the other common reagents used in the allergen-specific IgE portion of the assay.
Quantitative IgE antibody assays need a dual QC system, one that quality controls the total IgE calibration portion (A) and the second that quality controls the allergen specific portion (B):
To quality control the calibration portion of the assay, three QC sera containing known total serum IgE levels (high, medium, and low) should ideally be analyzed in each assay.
To quality control the allergen portion of the assay, each allergen-containing reagent should be considered already quality controlled by the manufacturer.
Thus, verification of the allergen reagent portion of the assay may be accomplished by analyzing a number of sera with known IgE antibody levels from 5 up to 15 different allergen specificities that are rotated among the different runs.
External quality management of IgE assays
Laboratories performing total serum IgE and allergen-specific IgE antibody measurements have to demonstrate satisfactory performance in external inter-laboratory proficiency testing surveys.
General principles
The primary goal of proficiency testing is to verify that all clinical laboratories accurately measure total serum IgE and correctly identify sera that contain IgE antibody of differing allergen specificities.
Total serum IgE results in kU/L (IU/mL) are compared to peer group means and ranges, and outlier laboratories are identified. Peer groups are defined by the assay method.
When evaluating the allergen-specific IgE antibody results, surveys collate two types of allergen-specific IgE antibody data for each respective assay method:
qualitative results (positive/negative) that reflect the presence, and in some cases, the relative level of IgE antibodies from semi-quantitative assays such as in multi-allergen screen; and
unit results (i.e. kUA/L) that are produced by quantitative IgE antibody assays based on heterologous calibration.
Outlier laboratories can be identified when a laboratory detects IgE antibody in a serum from a non-atopic person (clinical history negative, negative skin test result). Likewise, an outlier laboratory can be identified when an undetectable IgE antibody result is reported for a serum from an atopic individual (positive clinical history and a positive skin test and or provocation test). Moreover, with sufficient numbers of laboratories (n>10) providing quantitative measurements of allergen-specific IgE antibody, it is possible to determine if any single laboratory result is within or outside i.e. the 95% peer group range.
A survey should challenge the laboratory with at least two (i.e. Germany) to three (i.e. USA) cycles per year. Each cycle should involve the analysis of up to 6 sera for a total IgE and up to five allergen-specific IgEs. In addition, manufacturers of allergen-specific IgE assay reagents may offer their own quality assurance program (i.e. Quality Club®, Phadia ThermoFisher) for laboratories which purchase their reagents. The latter manufacturer conducted quality control programs are useful, but not viewed as sufficiently independent to serve as an impartial measure of laboratory performance for laboratory inspectors.
Proficiency testing in Germany (German speaking countries)
Two institutions offer proficiency surveys for total and allergen-specific IgE measurements in Germany, but also accept customers from abroad:
Reference Institute for Bioanalytics (RfB, https://www.rfb.bio/cgi/surveys) and
Society for Promoting Quality Assurance in Medical Laboratories e. V. (INSTAND e. V., http://www.instandev.de/en.html)
The RfB provides serum samples four times per year (Table 4). The evaluation of each survey results are listed for each method and graphically presented (https://www.rfb.bio/cgi/switchLang?lang=en): Quantitative total IgE results are plotted together (Figure 4), allergen-specific IgE results are illustrated separately for each allergen specificity and assay type (Figure 5).
External proficiency surveys for diagnostic allergy laboratories (modified from Hamilton et al., CLSI document I/LA20-A3, 2016).
| RfBa survey allergology, GE | INSTANDb e.V. allergy diagnostic, GE | UK NEQUAS,c UK | CAP-SE,d USA | |
|---|---|---|---|---|
| Variables | ||||
| Cycles per year | 4 (2 mandatory in GE) | 4 (2 mandatory in GE) | 6 | 3 (every 17 weeks) |
| Sera per cycle | 2 (individual or pools) | 5 (individual or pools) | 2 | 5 (individual or pools) |
| Multiallergen IgE antibody screen per serum | – | – | 1 | |
| IgE antibody measurements per serum | 6 (rotating important allergen sources from inhalant allergens, food allergens, hymenoptera venoms) | 6 (rotating important allergen sources from inhalant allergens, food allergens, hymenoptera venoms) | 4 (rotating important allergen sources from inhalant allergens, food allergens, hymenoptera venoms) | 5 (mixed positive/negative) |
| Allergen-specific IgE analyses per year per laboratory | No requirements | No requirements | 75 | |
| Total IgE analyses/year | No requirements | No requirements | No requirements | 15 |
| Reporting units: total IgE allergen-specific IgE | kU/L kUA/L and allergen class | kU/L kUA/L and allergen class | kU/L kUA/L and grade | kIU/L kIUa/L |
| Definition of outliers total IgE allergen-specific IgE | Variation exceeds ±30% | Variation exceeds ±30% | Method consensus | |
aReferenzinstitut für Bioanalytik (Reference Institute for Bioanalytics); https://www.rfb.bio/. bGesellschaft zur Förderung der Qualitätssicherung in medizinischen Laboratorien e. V. (Society for Promoting Quality Assurance in Medical Laboratories); http://www.instandev.de/en/eqas.html. cUnited Kingdom National External Quality Assessment Service; http://www.ukneqas.org.uk. dCollege of American Pathologists’ Diagnostic Allergy Survey; http://www.cap.org.
Illustration of total IgE results after proficiency survey allergology 4/2015 (RfB, Reference Institute for Bioanalytics; https://www.rfb.bio/): Plotted results of two different total IgE samples (left plot, x-axis: sample A, y-axis: sample B) and separated evaluation according to the type of assay kit (right listing and plot).
Illustration of allergen-specific IgE results to four different allergen specificities after proficiency survey allergology 4/2015 (RfB, Reference Institute for Bioanalytics https://www.rfb.bio/): histograms with allergen-specific IgE values and semi-quantitative classes.
Each histogram shows the frequency (y-axis) and distribution (x-axis) of allergen-specific IgE-results grouped according to the diagnostic method from different manufacturers (different lines).
Considerable variations appear between different total IgE assays as well as between laboratories using identical methods (Figure 4). More extensive variability is evident with the quantitative allergen-specific IgE results (Figure 5): Striking differences appear between various assay types as well as between laboratories using the same assay (detailed example of grass pollen specific IgE assay results in Figure 6).
Detailed Illustration of allergen-specific IgE results (sample C, left panel, sample D right panel) to timothy grass (g6) (Phleum pratense): histograms with stretches of linear allergen-specific IgE scales and color bar coding representing semi-quantitative classes.
The patterns (clustering of results around different medians) seem to indicate systematic discrepancies related to the assay design from different manufacturers [32] as well as performance errors with considerable variations between different laboratories applying identical methods (spreading of the results obtained with identical IgE-tests). These findings illustrate the urgent need of general harmonization (between IgE assays) and further improvement in laboratory performance for those assaying IgE samples. In contrast, INSTAND provides their proficiency survey results only to its customers; comparative long-term data have been prepared for publication [33].
Proficiency testing in Europe
Additional European and international sites providing proficiency trials are listed in the Eptis database (https://www.eptis.bam.de/eptis/WebSearch). A European study in several countries was performed in 1992, involving the UK, Belgium and the Netherlands [34].
Examples of other external quality assessment schemes are i.e.
the UK NEQAS (Immunology, Immunochemistry and Allergy) (http://www.immqas.org.uk), run from Sheffield Teaching Hospital,
the IQ-3 for specific IgE provided by the GECLID-SEI (External Quality for Diagnostic Immunology Laboratories under the auspices of the Spanish Society for Immunology), run in Spain (http://www.geclidsei.uva.es/)
the Australian RCPA Quality Assurance Program in Immunology (allergy specialty) (http://www.rcpaqap.com.au).
Proficiency testing in the USA
One well-subscribed program available internationally is the USA-based Diagnostic Allergy (SE) survey conducted by the College of American Pathologists (www.cap.org) (Table 4). In addition to providing a challenge for total serum IgE, it also provides five allergen-specific IgE antibody challenges per serum every 17 weeks (three challenges per year). In this survey, there is a sixth wild card specimen that is analyzed in the first and third cycles to allow specific IgE antibody measurements to rarer allergen specificities to be tested. In addition, some states in the United States such as New York also run their own external proficiency program for human immunoglobulins which includes total serum IgE. Outlier laboratories are identified that i.e. exceed the 99% confidence limit for their peer group. Figures 7 and 8 display representative total and allergen-specific IgE antibody levels as measured by North American laboratories in masked 2015 challenge sera. These data confirm that, with the exception of one method (see Figure 7), there is agreement in the total serum IgE levels reported from the multiple assays used (e.g. inter-assay CVs <15%). This is due in large part to the use of a common WHO human IgE IRP. In contrast, Figure 8 demonstrates wide divergence in the allergen-specific IgE antibody levels in kUA/L as reported by the three principal autoanalyzers used in North America. While reported IgE anti-peanut levels for this challenge specimen agree with each other, quantitative levels of IgE antibodies to the other allergen specificities vary widely, with no particular pattern. This variability is in large part thought to be related to heterogeneity of the composition of the assays’ allergen-containing reagents.
Total serum IgE levels (mean±1 SD) in kU/L as measured in 2 (SE-06 and SE-07) representative 2015 challenge specimens submitted to North American clinical immunology laboratories that perform diagnostic allergy testing.
The results are provided for five total serum IgE assays where more than 10 laboratories provided results so statistics could be performed. Data are presented for the following methods: Hycor EIA (H; n=20), Thermofisher Scientific Phadia ImmunoCAP (IC; n=199), Roche Cobas e600/E170 (C; n=21), Siemena ADVIA Centaur/XP (AC; n=19), Siemens Immulite 2000/XPi (IM; n=55). ALL=all methods combined generated inter-method coefficients of variation of <15%. These data demonstrate that total IgE assays when calibrated to the WHO IgE International Reference Preparation (with the possible exception of method H) measure comparable levels of IgE in human serum.
Allergen-specific IgE antibody levels (mean±2 SD; presented on a log scale) in kUA/L as measured in SE B 2015 challenge sera by North American laboratories that perform diagnostic allergy testing.
The results are provided for three IgE antibody assays where more than 10 laboratories provided results so statistics could be performed. Data are presented for the following methods (in order) for each allergen specificity: Hycor EIA (n=14), Thermofisher Scientific Phadia ImmunoCAP (n=259) and Siemens Immulite 2000/XPi (n=75). These data demonstrate that allergen-specific IgE assays as measured by the three principal autoanalyzers used in North American clinical immunology laboratories tend to report divergent quantitative IgE antibody results. There are the occasional specificities where IgE antibody results can agree (e.g. peanut). The magnitude of the inter-assay differences depends upon largely on the heterogeneity of the donor’s IgE antibody response to a particular allergen specificity.
Challenges for quality control involving new developments in allergen-specific IgE detection
Molecular allergology: from extracts to molecules
Naturally purified or recombinant proteins representing single allergens are being increasingly introduced into clinical allergen-specific IgE testing [35]. This could potentially lead to more uniformity between different assays formats since it will minimize the variation that occurs with allergen extracts. However, single allergens can present unique issues, as various isoforms occur that display heterogeneous IgE binding patterns which depend on correct folding and full presentation of potential IgE-binding epitopes. Moreover, due to licensing restrictions, not all IgE antibody manufacturers can supply the same component allergen reagents and thus external quality control data can become restricted to a limited number of different assays. Thus, differences between assay formats are still expected to occur when using single molecules. So far, only few studies have compared IgE antibody assay results that have investigated single molecules using assays from different manufacturers. However, identical single allergenic molecules might provide an ideal challenge condition for inter-assay comparisons as it (in theory) eliminates the variability of the allergenic reagent. Exchange of reagents should therefore be promoted in the interest of establishing more uniform results between different manufacturers of modern IgE-detecting assay platforms.
Singleplex and multiplex: from targeted testing to broad screening
Allergenic molecules are not only increasingly used for single determinations (singleplex-autoanalzyers) [9] but have also been utilized in micro-array chip based systems (i.e. ISAC112, Phadia/ThermoFisher) [15]. At present, 112 single allergens from 51 allergen sources (pollen, mites, animals, molds, food allergens and others) are offered for simultaneous IgE-testing (multiplex) using minute amounts of serum. These types of assays (not one, but rather i.e. 112 simultaneous tests) introduce new challenges for proper quality control, since only a small number of many allergen specificities present in the multiplex array can successfully be evaluated in internal or external quality management programs. The burden for the manufacturer will ultimately increase for providing proper technical evaluations for each of the applied single allergens. These results should be published [15] or at least be publicly accessible to allow proper information on assay performance characteristics of these new assay formats.
Future challenges
Proper quantitation: harmonizing heterologous IgE units
One of the unresolved problems relates to the quantitation of allergen-specific IgE. IRPs or other available standards for allergen-specific IgE do not exist and will, if ever available, only be developed for few selected allergen (sources). Subsequently, heterologous interpolation from total IgE (kU/L) to allergen-specific IgE (kUA/L) units (Figure 3) will remain the standard calibration scheme for an extended period. Manufacturers should ensure, that these units (kU/L and kUA/L) are almost equivalent and can quantitatively be compared. This has only been demonstrated occasionally [36] and needs still to be shown for all quantitative IgE-assays.
As a consequence, allergen-specific IgE tests are sometimes regarded as not really quantitative (Figure 3, part A), at best semi-quantitative assays (see Figure 3, part B). In light of the wide spread variations of allergen-specific IgE levels that are seen in present external proficiency trials it is indeed difficult at present to make the case for a truly quantitative assay for allergen-specific IgE. However, for good reasons (Table 5) the goal should be valid quantitation with units equivalent to kU/L interpolated from IRP for total serum IgE.
Summation for quantitative allergen-specific IgE assays with improved result quality.
| PRO |
| – Heterologous interpolation allows proper estimation of allergen-specific IgE values |
| – Allergen-specific IgE/total IgE-ratio is important for proper interpretation |
| – Modern IgE-tests should demonstrate an advanced analytical sensitivity (limit of quantitation: 0.1 kUA/L) |
| CONTRA |
| – Comparability of total IgE (kU/L) and allergen-specific IgE (kUA/L) units has not yet been demonstrated for most of the current assay systems |
| – Manufacturer-specific differences between quantitative allergen-specific IgE values still exist |
| – Wide spread of detected allergen-specific IgE concentrations at present in proficiency trials even in case of identical assays systems |
Improved reagents: role of allergen standardization and potential broad accessibility of allergen reagents
The allergen reagent plays an essential role for a valid detection of sIgE and its importance is often underestimated. Due to varying physicochemical properties and abundance of the allergenic proteins in the source material with its matrix, appropriate extraction methods are required to ensure the integrity, sufficient amount and binding capacity of single allergens in the final allergen reagent. Several steps could potentially harmonize the discrepancies between the sIgE results employing assays with their own allergen reagents from different manufacturers. However, due to the mutual interest of developers of commercial diagnostic tests it is unlikely that these measures will present a realistic goal for the upcoming future. Joint efforts by the majority of sIgE test developers would be necessary to agree and develop such common reagents, more specifically allergen extracts (standard preparations) as well as allergenic molecules. This could be obtained at least for the most prevalent allergen sources, which should finally replace the present individual allergen reagents from individual manufacturers.
Author contributions: All the authors have accepted responsibility for the entire content of this submitted manuscript and approved submission.
Research funding: None declared.
Employment or leadership: None declared.
Honorarium: None declared.
Competing interests: The funding organization(s) played no role in the study design; in the collection, analysis, and interpretation of data; in the writing of the report; or in the decision to submit the report for publication.
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©2016 by De Gruyter
Artikel in diesem Heft
- Frontmatter
- Allergie und Autoimmunität/Allergy and Autoimmunity / Redaktion: U. Sack/K. Conrad
- Qualitätskontrolle und Validierung in der diagnostischen Durchflusszytometrie
- Quality management in IgE-based allergy diagnostics
- Labordiagnostik bei systemischen Autoimmunerkrankungen
- Geriatrisches Labor/Geriatrics Laboratory / Redaktion: P. Schuff-Werner
- Surrogatmarker der Insulinresistenz bei Studienteilnehmern mit metabolischem Syndrom – Daten der Berliner Altersstudie II
- Neurologisches Labor/Neurological Laboratory / Edited by: H. Tumani/U.K. Zettl
- Expression of interferon type-I receptor isoforms, clinical response and development of neutralizing antibodies in multiple sclerosis patients – results of a prospective study
- Fallbericht/Case Report
- Labordiagnostik bei der monoklonalen Gammopathie unklarer Signifikanz (MGUS)
- Originalartikel/Original Articles
- Comparison between capillary glucose measured with a Contour glucometer and plasma glucose in a population survey
- Supplements to a recent proposal for permissible uncertainty of measurements in laboratory medicine
- Butyrylcholinesterase as an additional marker in the diagnostic network of acute myocardial infarction
- Buchbesprechung/Book Review
- Molekulare Allergiediagnostik
Artikel in diesem Heft
- Frontmatter
- Allergie und Autoimmunität/Allergy and Autoimmunity / Redaktion: U. Sack/K. Conrad
- Qualitätskontrolle und Validierung in der diagnostischen Durchflusszytometrie
- Quality management in IgE-based allergy diagnostics
- Labordiagnostik bei systemischen Autoimmunerkrankungen
- Geriatrisches Labor/Geriatrics Laboratory / Redaktion: P. Schuff-Werner
- Surrogatmarker der Insulinresistenz bei Studienteilnehmern mit metabolischem Syndrom – Daten der Berliner Altersstudie II
- Neurologisches Labor/Neurological Laboratory / Edited by: H. Tumani/U.K. Zettl
- Expression of interferon type-I receptor isoforms, clinical response and development of neutralizing antibodies in multiple sclerosis patients – results of a prospective study
- Fallbericht/Case Report
- Labordiagnostik bei der monoklonalen Gammopathie unklarer Signifikanz (MGUS)
- Originalartikel/Original Articles
- Comparison between capillary glucose measured with a Contour glucometer and plasma glucose in a population survey
- Supplements to a recent proposal for permissible uncertainty of measurements in laboratory medicine
- Butyrylcholinesterase as an additional marker in the diagnostic network of acute myocardial infarction
- Buchbesprechung/Book Review
- Molekulare Allergiediagnostik
