ALLERGOL. ET IMMUNOPATHOL., 1998;26(5):223-227
ORIGINAL ARTICLES
Determination of total and specific IgE using UNICAP 100: comparative study with the CAP system
P. Cots*, J. M. Pena**, J. Botey*, J. L. Eseverri*, A. Marín* and R. Ras**
*The Allergy and Paediatric Clinical Immunology Department. **The Paediatric Clinical Biochemistry Department. Maternity and Children''s University Hospital.
Vall d''Hebrón. Barcelona.
SUMMARY
The methods of serum determination of specific IgE to different allergens have showed lower diagnostic sensitivity than the alternative in vivo methods, the skin tests. The CAP system from Pharmacia, owing to its solid phase, has ameliorated this disadvantage, showing in various studies greater diagnostic sensitivity than the classic RAST, without affecting specificity. However, this system is still semi-automatic and requires daily calibration. UNICAP 100 is a completely automatic autoanalyser for total IgE, specific IgE and Eosinophil Cationic Protein, which combines the high sensitivity of the CAP system with complete automation and monthly calibration. The aim of the present study is to assess the practicality and reliability of UNICAP 100, when compared to the CAP System, for the determination of total IgE and specific IgE, as well as the sensitivity and diagnostic specificity; using as a reference skin tests in 150 paedriatic patients. The coefficients of variation in the study of intraseries imprecision ranged between 2.1% and 3.6% for total IgE and between 2.2% and 5.1% for specific IgE, depending on the allergen and the level studied. The intraseries imprecision ranged between 3.3% and 7.7% for total IgE and between 5.2% and 8.9% for specific IgE. The coefficients of correlation of the study of interchangeability with the results of the CAP System varied between 0.985 and 0.998, all the allergens tested (9) being interchangeable. Finally, the diagnostic sensitivity varied between 70% and 95% and the specificity between 87% and 100%. In conclusion, UNICAP 100 showed results that were interchangeable with the CAP System, noticeably improving the benefits owing to its complete automation and its calibration system.
Key words: Serum determination. Allergens. Diagnostic sensitivity.
Allergol et Immunopathol 1998;26:223-7.
INTRODUCTION
The discovery of IgE in 1967 by ishizaka (1) and Johansson (2) provided a scientific basis for allergy. The subsequent development of anin vitro test for its detection by Wide et al. in 1987 (3), commercialized as Phadebas RAST (RAST), was a significant advance in the diagnosis of allergic illnesses, as the determination of total and specific IgE in serum is today the most importante in vitro technique in the diagnosis of the cause of type I hypersensitivity.
Throughout this time, the RAST method has been widely evaluated, and the conclusion has been reached that it has high specificity but low sensitivity, especially when compared with the in vivo tests such as skin tests. For this reason, a new generation of methods has arisen, which attempt to overcome this disadvantage. During the last decade, a new in vitro test was introduced for identifying total and specific IgE, called the CAP System (4). Its most important contribution is its solid phase, which consists of a cellulose polymer activated within a capsule (the ImmunoCAP), which can bind more proteins than the corresponding paper-disk and covered tubes. This provides favourable conditions for the reaction, with shorter diffusion distances, so that the majority of the reactions between the IgE and the allergen occur very rapidly.
A number of studies carried out to date show, in general, a greater sensitivity of the CAP System compared to the RAST, drawing closer to the results provided by skin tests and identifying more positive cases (5-8), although this depends to a large degree on the allergen tested (greater sensitivity in the case of inhalants and less in the case of foodstuffs), which indicates that each allergen has to be evaluated separately to determine its sensitivity.
Both the classic method (RAST) and the current CAP System have sufficient specificity and sensitivity to confirm the diagnosis in many cases, but they have significant limitations, such as the need for daily calibration for all the allergens tested, and the lack of complete automation.
UNICAP is a new autoanalyser for total and specific IgE, which combines complete automation with the high sensitivity of the CAP System, without requiring daily calibration.
The aim of this study is to compare individual correlation between the CAP System and UNICAP 100 for each of the most common allergens to be found in our area, and to assess the sensitivity and diagnostic specificity using skin tests as a reference.
MATERIAL AND METHODS
The group comprised 150 paedriatic patient between 3 and 12 years old, with a clinical history of and positive skin tests for some type of allergen (inhaled and/or foodstuff).
Clinical histories were taken for each patients and included, amongst other information, family and personal history of atopy, and the current reason for consultation: bronchial asthma, spasmodic cough, allergic rhinitis, urticaria or atopic dermatitis. In addition, a detailed description of the attacks was recorded, as well as the treatment administered and its effectiveness.
Skin Tests
These were carried out on all patients, studying the allergens which are most commonly found in our environment. A Morrow-Brown type lancet was used for carrying out the prick test on the upper part of the forearm.
The allergenic extracts used were standardized in biological units and diluted in a saline solution treated with glycerin. The positive control was 1% histamine and the negative control was 9% codeine phosphate.
The results were read after ten minutes and were considered to be positive when the diameter of the erythema was larger than or equal to that caused by the histamine.
Assessment of Specific IgE in serum
In each case the blood was extracted by venipuncture, the serum samples were obtained by cold centrifugation and were stored in aliquots at -60° C before being analyzed. The assessment of total and specific IgE in serum was carried out using the CAP System and the UNICAP 100.
CAP System
The CAP System is an in vitro technique which consists of a solid phase for measuring total and specific IgE in blood or in undiluted plasma. It can be carried out using the RIA technique or with fluorometry. For the purposes of this study the fluorometry method was used.
The system used a mixture of monoclonal and polyclonal antibodies labelled with iodine-125 or with beta-galactosidase, which generates fluorescence. For this reason the test can be used as radioimmunoassay or as a fluorometry test. Both have a high immunoreactivity, allowing a greater amplitude in the measurements compared to the rest of the existing tests for determination of IgE.
The results are expressed in Kilo-units per litre compared to the WHO IgE standard. The detection range is between 0.35 and 100 KU/l.
UNICAP 100
This is a new autoanalyser for total and specific IgE. It has similar characteristics to the CAP System, but also has the following advantages: complete automation and the lack of necessity for daily calibration. The analyser dispenses sera and reagents, washes, and reads and transmits the results automatically. The capacity of each series is 48 determinations and the total time taken is 2.5 hours.
Allergens tested
D. Pteronyssinus (d1)
Cat. epithelium (e1)
Alternaria (m6)
Phleum (g6)
Parietaria judaica (w21)
Olea europaea (t9)
Egg white (f1)
Peanut (f13)
Casein (f78)
RESULTS
Comparative precision of CAP and UNICAP
The precision characteristics studied tables I and II showed little variation using the UNICAP method compared to the AP System for the determination of total and specific IgE. Moreover, both had similar values regarding recuperation and detection, without cases of contamination.
Table I | |||||
Reliability parameters of CAP-UNICAP 100 | |||||
| CAP to | UNICAP to | CAP E | UNICAP E | ||
| CV Intra% | 3-5 | 2-4 | 4-60 | 2-5 | |
| CV Inter % | 6-9 | 3-8 | 7-11 | 5-9 | |
| Table II | ||||
| Reliability parameters of CAP-UNICAP 100 | ||||
| CAP to | UNICAP to | CAP E | UNICAP E | |
| Contam | -0.007 | -0.002 | -0.008 | 0.001 |
| Detecc | 6 | 4 | 0.2 | 0.15 |
| Recuper | 98 | 101 | 97 | 96 |
| CAP to: CAP total, UNICAP to: UNICAP total, CAP E: CAP especific, | ||||
| UNICAP E: UNICAP specific. | ||||
CAP-UNICAP correlation
The coefficient of correlation found between the various allergens varied between 0.98 and 0.99. The slope of the regression line varied between 1.26 for casein (f78) and 0.93 for Alternaria (m6) (table III).
| Table III | |||
| Regression and correlation between CAP and UNICAP 100 in patients with positive skin test results | |||
| Y (UNICAP) = aX (CAP) + b | |||
| Allergen | r | a | b |
| d1v | 0.998 | 1.12 | -0.73 |
| g6 | 0.995 | 0.98 | 0.34 |
| m6 | 0.994 | 0.93 | 0.05 |
| e1 | 0.985 | 1.10 | -0.44 |
| w21 | 0.995 | 1.02 | 0.52 |
| f1 | 0.988 | 0.97 | 0.18 |
| f13 | 0.997 | 1.01 | 0.01 |
| f78 | 0.992 | 1.26 | -0.37 |
| d1: D. Pteronyssinus; e1: epitelio de gato; m6: Alternaria; g6: Phleum; | |||
| w21: parietaria judaica; t9: Olea europea; f1: clara de huevo; f13: cacahuete; f78: caseína. | |||
Sensitivity (s) and specificity (E) diagnosis of CAP and UNICAP 100
The S diagnosis for the UNICAP 100 method did not show significant variations compared to CAP for the various allergens tested. The S of CAP varied between 75% for casein (f78) and 93% for-D. Pteronyssinus (d1). The S of UNICAP varied between 70% for casein (f78) and 95% for-D. Pteronyssinus (d1) (table IV).
| Table IV | ||
| Sensitivity of CAP and UNICAP 100 compared to skin test | ||
| Sensitivity (%) | ||
| Allergen | CAP | UNICAP 100 |
| d1 | 93 | 95 |
| e1 | 92 | 90 |
| m6 | 85 | 89 |
| t9 | 90 | 82 |
| w21 | 90 | 93 |
| g6 | 89 | 88 |
| f1 | 83 | 80 |
| f13 | 91 | 92 |
| f78 | 75 | 70 |
| Total inhalants | 95 | 94 |
| Total food allergens | 84 | 83 |
| d1: D. Pteronyssinus; e1: epitelio de gato; m6: Alternaria; g6: Phleum; | ||
| w21: parietaria judaica; t9: olea europea; f1: clara de huevo; f13: cacahuete; f78: caseína. | ||
There were practically no variations observed with respect to the S of inhaled allergens (95% CAP and 94% UNICAP), nor those of foodstuffs (84% CAP and 83% UNICAP).
Similarly, the E diagnosis using each method did not show great differences. The E of CAP varied between 82% for peanut (f13) and 100% for olive (t9). The E of UNICAP varied between 88% for peanut (f13) and 100% for casein (f78) (table V).
| Table V | ||
| Sensitivity of CAP and UNICAP 100 compared to skin test | ||
| Specificity (%) | ||
| Allergen | CAP | UNICAP 100 |
| d1 | 85 | 87 |
| e1 | 91 | 96 |
| m6 | 92 | 98 |
| t9 | 100 | 92 |
| w21 | 93 | 98 |
| g6 | 88 | 90 |
| f1 | 90 | 95 |
| f13 | 82 | 86 |
| f78 | 91 | 100 |
| Total inhalants | 98 | 93 |
| Total food allergens | 93 | 96 |
| d1: D. Pteronyssinus; e1: epitelio de gato; m6: Alternaria; g6: Phleum; | ||
| w21: parietaria judaica; t9: olea europea; f1: clara de huevo; f13: cacahuete; f78: caseína. | ||
With respect to the E for inhaled allergens, this was slightly higher for CAP (98% CAP and 93% UNICAP), with the opposite being true for the foodstuff allergens (93% CAP and 96% UNICAP).
DISCUSSION
Since in 1967 Wide et al. discovered RAST, as a first trial in detecting specific IgE in the blood of allergic patients, a great variety of systems have improved the technique, obtaining completely validated in vitro tests for the determination of total and specific IgE in serum. When the determinations are carried out correctly and interpreted in conjunction with a clinical history and a physical examination, they represent a basic and confirmatory complement to the skin tests. The in vitro tests have some advantages compared to the skin tests: the results are not influenced by the farmacotherapy, the reagents are stable and have a long life before expiry, there is no risk of systemic reaction, and finally, they show less variability between different laboratories.
The prototype of in vitro analysis, the RAST system, is less sensitive than the in vivo tests. For this reason, in recent years a number of methods have been designed which have attempted to ameliorate this disadvantage, among them the CAP System.
The CAP System is a new method developed by Pharmacia for measuring total and specific IgE in serum, and is currently the most advanced in vitro test for the diagnosis of type I hypersensitivity. There appears to be a good correlation between RAST and the CAP system, with similar or better results when the two techniques are compared to detect specific IgE. This study was assessed at our Unit by R. Alonso et al. in 1995 on a paedriatic population, with coefficients of correlation CAP-RAST of the various allergens tested of between 0.971 and 0.991, and an increase in diagnostic sensitivity for all the allergens studied with the CAP System, without the specificity changing.
Therefore the CAP System is considered to be the preferred in vitro test for carrying out adequate clinical, diagnostic and therapeutic monitoring in patientes with an allergic pathology, offering a high level of sensitivity and specificity.
However, despite these advantages, this method is semi-automatic and requires daily calibration for the allergens tested. For this reason a new autoanalyser has been studied for total IgE, specific IgE and Eosinophil Cationic Protein, called UNICAP 100, which combines complete automation with the high level of sensitivity of the CAP System. According to the results of our study, there is a good correlation between the CAP System and UNICAP 100, without significant variations regarding sensitivity and specificity of the allergens tested, using skin tests as a reference.
In conclusion, the CAP System is the most frequently used laboratory method for the diagnosis of allergic illnesses mediated by IgE. If one adds to the results obtained in our study the advantages of the new method, such as the complete automation and the lack of requirement for daily calibration, UNICAP 100 appears as a significant advance among the laboratory tests currently used.
RESUMEN
Los métodos de determinación sérica de IgE específica a diferentes alergenos han demostrado menor sensibilidad diagnóstica que su alternativa in vivo, las pruebas cutáneas. El sistema CAP de Pharmacia, debido a su fase sólida, ha mejorado este inconveniente presentando en diversos estudios mayor sensibilidad diagnóstica que el clásico RAST, sin modificar su especificidad. Este sistema todavía es semiautomático y con calibración diaria. UNICAP 100 es un autoanalizador totalmente automático de IgE total, IgE específica y proteína catiónica del eosinófilo que combina la alta sensibilidad del sistema CAP con automatización total y calibración mensual. El objetivo del presente estudio es conocer la practicabilidad y fiabilidad del UNICAP 100 en paralelo con CAP System para la determinación de IgE total y específica, así como la sensibilidad y especificidad diagnóstica utilizando como referencia las pruebas cutáneas en 150 pacientes pediátricos. Los coeficientes de variación del estudio de imprecisión intraserial oscilan entre 2,1% y 3,6% para IgE total y entre 2,2% y 5,1% para IgE específica, según el alergeno y nivel estudiado. La imprecisión interserial oscila entre 3,3% y 7,7% para IgE total y entre 5,2% y 8,9% para IgE específica. Los coeficientes de correlación del estudio de intercambiabilidad con los resultados de CAP System oscilan entre 0,985 y 0,998, siendo todos los alergenos ensayados (9) intercambiables. Finalmente la sensibilidad diagnóstica oscila entre 70 y 95% y la especificidad entre 87 y 100%. En conclusión UNICAP 100 presenta resultados intercambiables con CAP System mejorando sensiblemente las prestaciones por su automatización total y su sistema de calibración.
Palabras clave: Determinación sérica. Alergenos. Sensibilidad diagnóstica.
REFERENCES
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Correspondence:
J. Botey
Diagonal, 347 entlo.
08037 Barcelona
