Inter‐laboratory comparison of semiquantitative allergen‐specific Immunoglobulin E test: 7 years of experience in Korea

Abstract Introduction Multiple allergen simultaneous test (MAST) is widely used as a screening tool for allergic diseases and has the advantage of providing specific IgE (sIgE) results for various allergens in semiquantitative class. We have continuously conducted external quality assessment (EQA) since 2012 for clinical laboratories performing MAST using AdvanSure allergy screen test (LG CHEM, Korea). This study provides an account of the EQA experience. Methods Samples were prepared using pooled sera collected from patients with suspected allergic disease and sent to each laboratory twice a year. Each round included 4–6 serum samples with sIgE for 10–20 inhaled or food allergens. The acceptable class value was the most frequently reported MAST class ±1 titer that exceeded 80% of the total laboratory results. Results The average number of participating laboratories was 76 (49–90) and the average response rate was 97.3% during the entire survey period. The acceptable rates were consistently high at 97.7% ± 3.7%. Of the total 537 trials, 18 trials (3.4%) were regarded as nonconsensus results, in which acceptable answers did not exceed 80%. For unacceptable results, the false‐negative rate (1.5% ± 2.8%) was higher than the false‐positive rate (0.8% ± 2.7%) (p < 0.001). MAST class results were correlated with quantitative IgE results by ImmunoCAP (Spearman's correlation coefficient of 0.682 (p < 0.001) and gamma index of 0.777 (p < 0.001). Conclusion Although EQA for MAST showed a high level of acceptable answer, some allergen assays require harmonization. Continuous performance of systematic EQA is needed to improve the accuracy of sIgE assays and quality control in clinical laboratories.


| INTRODUC TI ON
The incidence of allergic conditions has continuously increased over the past decades. 1 Although in-vivo skin prick test (SPT) is available in clinical practice, some limitations could cause erroneous results in patients taking antihistamines or suffering skin diseases, among others. 2 Therefore, detection of serum allergen-specific Immunoglobulin E (sIgE) is essential for the diagnosis and management of IgE-mediated hypersensitivity reaction and allergic diseases. 3 Commercially available assays using the principles of enzyme immunoassay (EIA) or fluorescent enzyme immunoassay (FEIA) are now widely used. 4 The ImmunoCAP (Thermo Fisher Scientific) based on the FEIA technique is the most popular for the detection of sIgE for individual allergens. 5 However, when testing for multiple allergens is required, the cost is relatively high and labor-intensive compared to multiplex assay. 6 Currently, several multiplex allergen screening tests (MAST) have been developed and are widely used with the advantages of small sample size, short turnaround time, and cost-effectiveness for a various spectrum of sIgE testing. 2 Although MAST showed relatively low sensitivity and accuracy compared to ImmunoCAP assay, it is useful for screening the multiple sIgEs and provides the degree of sIgE measurement with semiquantitative class (from 0 to 6) results. 2,7 External quality assessment (EQA) is critical to ensure the accuracy and reliability of laboratory results. However, inter-laboratory proficiency testing for sIgE detection was not popular and was only recently introduced into the program of the Korean Association of External Quality Assessment service. The AdvanSure allergy screen test (LG CHEM, Ltd.) is a fully automated line immunoassay for semiquantitative detection of multiple sIgEs developed in Korea. 2,8 Since 2012, we have established local EQA programs for laboratories performing MAST using AdvanSure allergy screen tests. The purpose of this study is to review EQA experience by analyzing semiquantitative data for 7 years and to evaluate the performance of EQA for sIgE test.

| Preparation of serum samples
Leftover sera were collected from patients who visited Seoul St. Mary's hospital and requested the MAST assay in suspicion of allergic diseases.
We prepared pooled sera containing sIgE against a wide spectrum of inhalants or food allergens. Pooled sera were homogenized using an agitator at 180 rpm and then filtered using sterile filter paper. Sealed aliquots (each 650 µl) were stored at −80°C until proficiency testing.
This study was approved by the Institutional Review Board of Seoul St.

| External quality assessment
Twice a year, aliquots were sent to clinical laboratories in Korea where MAST was performed using AdvanSure allergy screen test. In each round, all participant laboratories received 4-6 serum samples requiring 10-20 sIgEs test results per serum. The laboratories were asked to test sIgE following the same procedure as for routine samples. Results were requested to be submitted in MAST classes. The sIgE classification scales were as follows; class 0: under 0.35 kU/L, class 1: 0.35-0.7 kU/L, class 2: 0.7-3.5 kU/L, class 3: 3.5-17.5 kU/L, class 4: 17.5-50 kU/L, class 5: 50-100 kU/L, class 6: over 100 kU/L. Class ≥1 was interpreted as positive.

| Data evaluation according to the acceptable class values
We analyzed the results received from individual laboratories, and provided overall results and acceptable class values for each allergen. The acceptable class values were defined the most frequently reported MAST class ±1 titer that exceeded 80% of the total laboratories according to the performance evaluation criteria of the Korean Association of External Quality Assessment (KEQAS) for semiquantitative tests. 9 If the sum of the most frequent class and +/− 1 grade was below 80%, the acceptable ranges was not determined and defined as a nonconsensus result. Nonconsensus results were excluded from further analysis. False-negative was when the response result was lower grade than the acceptable range, and false-positive was when the response was in a higher grade than the acceptable answers. For negative sera, any positive result was deemed false. We analyzed subgroups of foodstuff, pollen, mites, mold, and animal epithelia.

| Data comparison with quantitative ImmunoCAP
The concentrations of sIgE with the ImmunoCAP from the 9th round. ImmunoCAP system provided the results using a six-class system, as same as MAST, and values of 0.35 kUI/L or more (Class ≥1) were interpreted as positive. The semiquantitative comparisons were performed using the gamma index and Spearman's rank correlation analysis. 10

| sIgE EQA program
The average number of the participants was 76 with the maximum number in the 13th round (n = 90) and the minimum in the 1st round (n = 49). The average response rates increased from 89.8% to 100% (97.3% ± 3.1%) during the survey period ( Figure 1). The average accept rates were consistently high (97.6% ± 1.0%) over the period. The survey materials consisted of 76 foodstuff, pollen, mites, mold, and animal epithelia allergens with varying intensities from negative to strongly positive (Table 1). During the entire period, the most frequently participated allergens were D. farinae (24 trials) and D. pteronyssinus (24 trials) including samples with varying results of IgE ranging from negative to class 6. The allergens of animal epithelium consisting cat, dog, and sheep were included in a total of 35 trials. Thirty-eight foodstuffs were included in a total of 206 trials. Six allergens for mites were included in 92 trials. The 5 mold allergens and 24 pollen allergens were included in 42 and 162 trials, respectively.

| Analysis of sIgE EQA results in trials with consensus results
Of the total 537 trials, 18 trials (3.4%) were regarded as nonconsensus results, in which the percentage of acceptable answers did not exceed 80% of the total number of participants. In a total of 519 allergen trials with consensus results, an average of 97.7% of the participants presented acceptable results ( Table 2). The average rate of false-negative results (1.5% ± 2.8%) was higher than that of falsepositive results (0.8% ± 2.7%) (p < 0.001). Next, we analyzed the participants' results according to allergen subgroups, which included foodstuffs (n = 199), pollen (n = 160), mites (n = 84), mold (n = 41), and animal epithelia (n = 35). There were no significant differences in acceptable rates in each allergen subgroup, and all allergen groups showed false-negative results more than false-positive results.
Broad acceptable answer reporting rates of >5% standard deviation (SD) were found for shrimp (5.3%), tuna (5.5%), Japanese hop (5.5%), and Russian thistle (5.9%). Figure 3 shows the reporting rate of unacceptable results for each allergen by dividing it into false positive and false negative. Among food allergens, mackerel, salmon, and tuna showed only false positive answers.

| Analysis of sIgE EQA results for allergens with nonconsensus results
The distribution of reporting results for allergens with nonconsensus result are shown in Table 3

| Comparison of sIgE EQA results with ImmunoCAP results
Semiquantitative comparisons between ImmunoCAP and MAST are shown in Table 4. Within the total allergens available for comparison, spearman's correlation coefficient was 0.682 (p < 0.001) and the gamma index was 0.777 (p < 0.001). In allergen subgroups, foodstuffs (high positive), mites (moderate positive), mold (moderately positive), and pollen showed significant associations between the IgE class results of MAST and ImmunoCAP.

| DISCUSS ION
Until participating in KEQAS in 2020, there was no EQA program for sIgE assay in Korea. In this study, we reviewed EQA experience with interlaboratory comparisons of allergen-specific IgE assays conducted by a single institution since 2012. During the entire survey program across 14 round, relatively high rates of acceptable answer reporting were found. However, some allergens showed differences in IgE results, making it impossible to determine the consensus even using the same reagents. Therefore, we confirmed the need for a systematic EQA to improve the accuracy of assay and the quality control of clinical laboratories.
The performance evaluation criteria for semiquantitative and qualitative IgE tests have not yet been standardized. 12 In this study, EQA for semiquantitative IgE results was analyzed by applying the performance evaluation criteria of KEQAS. 9 Koch and Aberer analyzed the EQA of the Austrian allergy test for 25 years with an acceptable range of ±1 or 2 of the most frequent values. Also, for a positive sample, a case where the answer was higher than the acceptable answer by grade 2 or negative, and for a negative sample reported as positive was defined as unacceptable. 13

No. of trials Frequency (%) of participants' results in each trial
Acceptable results This study is meaningful as the EQA program was conducted for domestically developed reagent. Due to the significant differences in the prevalence of allergic disease by age, gender, and ethnicity, the allergen panels should be modified following the geographical region and target population. [15][16][17] The changes in environmental materials in modern society must be taken into that exceeded 80% of the total laboratories. Class results that contain more than 80% of the sum of all results are shown in dark red.

TA B L E 3 (Continued)
account for the progressive development of the MAST assay. 18 Also, the strength of this EQA program is its voluntary participation in laboratories using the same products and continuously collecting national data for 7 years. As expected of high compa- Therefore, laboratories performing sIgE assay require regular calibration and internal/external quality control, or interlaboratory comparison.
In this study, 7 years of IgE EQA experience was reviewed and the reported results were analyzed. Our experience in sample manufacturing, shipping, and reporting results analysis is expected to assist in the successful implementation of other IgE EQA programs.
Agreed regional or national consensus may help harmonize laboratory diagnostic strategies and explain the need for EQA along with strengthening education on their use.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data that support the findings of this study are available from the corresponding author upon reasonable request.