Detection of Specific IgE Patterns in Egyptian Atopic Children

Transcription

1 Research Journal of Medicine and Medical Sciences, 4(2): , , INSInet Publication Detection of Specific IgE Patterns in Egyptian Atopic Children Nehal Salah Hasan, Mona Abd El Kader Awad, Mohamed Kadry El- Masry, Inas Ismail Raafat, Mai Mahmoud Sherif, Emad Eldin Ezzat Salama and Nevine El-Helaly 1 Department of Clinical and Chemical Pathology, National Research Centre, Department of Clinical and Chemical Pathology, Faculty of Medicine, Cairo University, 3 Department of Pediatrics, National Research Centre, 4 Department of Pediatrics, Faculty of Medicine, Cairo University, Cairo, Egypt 2 Abstract: Background: For the diagnosis of allergy, presence of allergen-specific immunoglobulin E (IgE) usually is established either by allergen skin tests or by in vitro allergen-specific IgE measurements. Aim: The aim of our study is to detect the pattern of allergen positivity in a sample of Egyptian atopic children. Also to evaluate the diagnostic and clinical performance of chemiluminescence technique in the diagnosis of inhalants and food allergies, through the detection of total and specific IgE and comparing it with the standard skin prick testing and patient clinical condition. Methods: The study included 40 atopic children aged between 3 years and 15 years with known history of allergic conditions, they were tested for presence of allergens by the gold standard in vivo skin pricks test and in vitro chemiluminescence technique. Results: Our results revealed that both techniques agreed that mite is the commonest allergens among Egyptian children. Conclusion: Chemiluminescent test is a sensitive quantification method as it fulfils many of the requirements for good evaluation being time saving, precise, with very low detection limits. Key words: Chemiluminescence - Specific IgE - Atopy. INTRODUCTION Allergy is defined as the tendency to develop adverse symptoms due to an immune response to normally innocuous substances. The prevalence of allergic diseases has increased considerably over the last years, particularly in the more developed regions of the world. As a result, it is rapidly becoming a major health problem, resulting in [1] significant economic losses. Genetic factors alone cannot explain the increasing prevalence, nor can they explain the large differences in prevalence among regions of similar ethnic backgrounds. Thus, environmental factors may also play a major role in the development and expression of allergic disease. Childhood infections, vaccination programs, and changes in life-style, include: indoor conditions, diet, and microbial gastrointestinal flora, have all been suggested as causes for the increasing [2] prevalence. A huge variety of agents found in the environment can provoke an allergic response. The most recognizable symptoms of allergy occur as the result of an immediate hypersensitivity reaction that can affect any of a number of tissues throughout the body. This reaction is mediated by allergen-specific IgE antibody that is attached to the surface of mast cells. When these cells encounter an allergen recognized by the IgE on their surface, they secrete compounds that produce allergic symptoms. These compounds include histamine, [3,4] tryptase and heparin. The identification of responsible allergens remains a key step for practicing allergen avoidance and specific immunotherapy. The diagnosis is mainly based upon an evocative clinical history (including temporal association between symptoms and allergen exposure), positive skin tests, which are considered the gold standard in this context. However, skin prick testing as a diagnostic test is made difficult especially in children by the lack of standardized antigen for testing and is contraindicated in some patients with history of life threatening anaphylaxis. In these cases, functional in vitro tests are necessary. The general concept of those tests is to mimic in vitro the contact between allergens [5] and the antibodies responsible for symptoms. The use of serological assays to measure specific IgE such as radioallergosorbent assay. (RAST) and enzyme linked immunosorbent assays (ELISA) have [6] different levels of sensitivity and specificity. Corresponding Author: Nehal Salah Hasan, Department of Clinical and Chemical Pathology National Research Centre (NRC), Cairo, Egypt Phone: Fax: nemyb2006@yahoo.com 475

2 The aim of our study is to detect the pattern of allergen positivity in a sample of Egyptian atopic children, also to evaluate the diagnostic and clinical performance of chemiluminescence technique in the diagnosis of inhalants and food allergies, through the detection of total and specific IgE and comparing it with the standard skin prick testing and patient clinical condition. MATERIALS AND METHODS The study included 40 atopic children aged between 3 years and 15 years with known history of allergic conditions such as allergic rhinitis, asthma, and atopic dermatitis. Patients were chosen from the allergy out clinic of the National Research Centre, Cairo University Children Hospital and Vacsera Centre between the periods of March 2007 and June Each patient was subjected to the following: 1- Thorough clinical history as regards allergic conditions. 2- Ten ml venous blood sample from each patient was withdrawn into a 10mL serum separator tube or red-top tube. Patient needed not be fasting. No special preparations were necessary. A blood sample was drawn from each child after parental consent to perform the following: 1. Estimation of total serum IgE and specific IgE by Enzyme Enhanced Chemiluminescence MAST CLA-Hitachi Chemical Diagnostics system using a large battery of different allergens, this panel of allergens are chosen to include the most common encountered inhalant and ingested allergens that has been grouped by geographical and clinical prevalence offering a wide variety for the [7] physicians in their management program. The panel used is the Taiwan Asian panel (as it was found to be the most matching panel to Egyptians) that includes 36 parameters: Total IgE, Housedust, Mite Farinae, Mite pterony, Cockroach Mix, Feather Mix, Dog, Cat, Candida, Aspergillus, Cladosporium, Penicillium, Alternaria, Grass Mix, Bremuda Grass, Wheat, Cod fish, Pork, Beef, Egg, Whole, Egg Yolk, Milk, Yeast Brewer, Soybean, Peanut, Vegetable Mix, Ragwd Mix II, Pine Mix, Cottonwd, Mulberry Mix, Pigweed Mix, Corn, Crab, Shellfish Mix, Shrimp, and Eucalyptus. Inside every Pette are up to 36 cellulose threads, each containing a selected allergen. The patient's blood sample is drawn and the serum is separated and aspirated into the device. IgE-specific antibodies in the serum bind into allergens in the threads that trigger an allergic reaction. Then additional reagents are added to the panel, which produces light in varying strengths proportionate to the intensity of the allergic response. The results are determined by loading the Pette into the CLA-1 Luminometer. Interpretation of the Results: To calculate the patient's IgE response, the instrument automatically subtracts the emission level of the Negative Blanking Control Thread from the emission level of each specific IgE thread. The CLA-1 Luminometer assigns class values from 0 to 4 to the amount of light emitted by threads in the MAST Pette. These values comprise the MAST class allergy scoring system of the MAST allergen- specific IgE assay. The amounts of IgE associated with MAST class values and instrument readings are listed in the (Table 1). 2. Skin testing: The panel used in skin testing includes 11 parameters: Mite mix, Cat pelt, Ashes, Timothy grass, Birch, Mesquite, Sagebruch, Lamb s quarter, Russian Thistle, Plantain English and Perennial rye. The manufacturer of allergens for skin prick tests is: Omega Laboratories Limited in Canada, Montreal, Quebec, they are distributor of Hollister-Stier products. Methodology of Skin Prick Test: 1. We made sure that all children did not receive any antihistaminic drugs, epinephrine, ketotifen or steroids at least 72 hours prior to the test. These medications are known to suppress skin test reaction. 2. The test was done on the volar surface of the forearm, avoiding the flexures. 3. The area was cleaned first with 70% ethyl alcohol and allowed to dry in air. 4. A droplet of each of the allergen extracts was placed on the skin, keeping one inch distance apart, in order to avoid overlap of the reactions. 5. The skin was pricked through the droplet at a sharp angle using a sterile lancet. This procedure should slightly abrade the skin, however, superficial enough to avoid drawing of blood. 6. The lancet was wiped between pricks using a sterile piece of cotton or tissue to avoid mixing of the allergenic extracts. 7. For each patient a negative control using physiologic saline and a positive control using histamine phosphate 1% was done. 8. The prick test sites were examined 15 minutes after pricking. A positive skin reaction was evident by a wheal and flare. If no reaction developed within 15 minutes, we had to wait for another 15 minutes and re-examine the test site before 476

3 considering the test to be negative. The degree of positivity of the immediate skin reaction corresponds to the size of the wheal obtained. 9. Positivity was graded as follows: +: wheal was 3-5 mm ++: wheal was 5-10 mm +++: wheal was 10-15mm ++++: wheal was > 15mm 10. Certain precautions were available as the skin tests were done: 1: aqueous adrenaline: first measure to be applied in treatment of a developing reaction. Antihistamines Corticosteroid Oxygen 11. The patients were observed for at least 30 minutes following any test. They were advised to ask immediate medical consultation if constitutional symptoms appeared. Statistical Analysis: Data was presented as numbers and percentage. Sensitivity which is the chance of testing positive among those with the condition, was calculated by the equation: True positive / (True positive + false negative). Specificity which is the chance of testing negative among those without the condition, was calculated by the equation: True negative / (True negative + false positive). Positive Predictive Value which is the chance of having the condition among those that test positive was calculated as: True positive / (True positive + False positive). Negative Predictive Value which is the chance of not having the condition among those that test negative was calculated as: True negative / (True negative + false negative). RESULTS AND DISCUSSION Results: In this study; house dust & mite are the highest percentages among the tested patients by using the CLA technique (Table 2), and these results did not differ than the skin prick test results (Table 3). The overall percent concordance between CLA & SPT techniques regarding the shared panels was 77.5%. It was found that there is a 100% agreement between the two techniques regarding the mite allergen, 55% agreement regarding the cat allergen and 77.5% agreement regarding the grass allergen (Table 4). The mite allergen came on the top of causative allergens causing bronchial asthma by 100%, while cat allergen gave the highest percentage among patients suffering from allergic rhinitis 46.15%. Although we had a small sample of atopic dermatitis patients; they were all allergic to peanut 100% (Table 5). Table 5 represents allergen positivity by the CLA technique among each of the 3 groups of patients, showing that house dust & mite are the highest percentages (100%) among the asthmatic tested patients, while allergic rhinitis patients showed positivity to large number of allergens with no peak to a certain allergen except for cat allergen (46.15%). Although we had a small sample of atopic dermatitis patients; they were all allergic to peanut (100%). When using the chemiluminescent technique for detection of sensitive allergens in children with atopic diseases in comparison to the gold standard skin prick test, it was found that the CLA technique has a sensitivity of 97.7% and specificity of 73.9%; a positive predictive value of 70% and a negative predictive value of 98.1% (figure 1). Discussion: Allergic diseases (asthma, atopic dermatitis, allergic rhinitis and food allergy) are the [8] commonest chronic diseases of childhood.the early identification of allergies and appropriate interventions are important to prevent progression to more significant disease. The use of objective diagnostic testing aids in implementing appropriate evidence-based medical [9] management. [10] These results were in agreement with who demonstrated that sensitization to D. pteronyssimus and cat were usually the most prevalent among the studied [11] allergens, and also with who reported that indoor allergens were the predominant sensitizing allergens detected on skin prick test as a measure of specific IgE in patients tested at King Abd El-Aziz University Hospital, particularly house dust mites. Our CLA results revealed that cat and dog came on the top of the list of sensitizing allergens with a percent of 65% and 62.5% respectively, this finding [12] was in accordance with who reported that sensitization to cat and dog are common even in subjects who claimed no direct exposure. Moreover, [13] proved that certain locations had levels of dog and cat dander above the threshold levels considered to be risk factors for both sensitization and symptoms, regardless of the presence of a pet in the home. In this study, the in vitro CLA test with 65% results and to a little extend the skin test with 20% results showed that the cat is one of the commonest sensitizing allergens, and [14] this agrees with who stated that allergens from cat are among the most potent elicitors of allergic disease. Using the in vitro CLA technique and not the SPT, peanut was also a common allergen with a percent of [15,16] 42.5% among all the patients, this agreed with who stated that among all food allergies, peanut allergy is the third most prevalent allergen occurring among young children and is the most common food allergy in older children, adolescents and adults. The allergenic proteins in peanut allergies are vicilin and conglutin. 477

4 Table 1: Amounts of IgE associated with MAST class values MAST Class Luminescence (LU) Allergen-Specific IgE Concentration 4 >242 Very High High Moderate Low 1/ Very Low Non detectable Table 2: The pattern of allergen positivity by the CLA technique Allergen Number Percent (%) Allergen Number Percent % Total IgE Beef 6 15 Housedust Egg, Whole Mite, farinae Egg, Yolk Mite, pterony Milk Cockroach Mix Yeast, Brewer Feather Mix Soybean Dog Peanut Cat Vegetable Mix 2 5 Candida Ragwd MixII Aspergillus Pine Mix Cladosporium Cottnwd, blk Penicillium Mulberry Mix Alternaria Pigweed Mix 2 5 Grass Mix Corn 2 5 Bremuda Grass 6 15 Crab 0 0 Wheat 6 15 Shellfish Mix Cod fish Shrimp 2 5 Pork 4 10 Eucalyptus Table 2 represents the pattern of allergen positivity by the CLA technique showing that house dust & mite are the highest percentages among the tested patients. Positivity was considered from MAST class 1/0 up to 4 CLA: Chemiluminescence MAST CLA-Hitachi Chemical Diagnostics system Table 3: The pattern of allergen positivity by the Skin prick test Allergen Number Percent (%) Mite Mix Cat pelt 8 20 Ashes 0 0 Timothy grass Birch

5 Table 3: Continue Mesquite 2 5 Sage bruch 0 0 Lamb's quarter 0 0 Russian thistle 0 0 Plantain English 0 0 Perennial rye 0 0 Table 3 represents the pattern of allergen positivity by the Skin prick test showing that mite mix is the highest percentage among the tested patients. SPT: Skin prick test Table 4: Concordance between CLA & SPT regarding the shared panels Allergen +ve agreement -ve agreement Percent Concordance % Overall Percent Concordance % Mite Cat Grass Table 4 shows the concordance between CLA & SPT techniques regarding the shared panels, where a 77.5% was the overall percent concordance between them. N.B: The comparison was done between the results of CLA & SPT for the same patient. Table 5: Allergen positivity in different allergic condition using chemiluminescent technique Allergen Bronchial asthma Allergic Rhinitis Atopic dermatitis N=23 cases(%) N=13 cases(%) N=4 cases(%) Housedust Mite, farinae Mite, pterony Dog Cat Aspergillus Candida Penicillium Cladosporium Alternaria Soybean Peanut Milk It has been found in our patients that there is a very high correlation and concordance between in vitro CLA and in vivo SPT results in the diagnosis of cases shown to be allergic to mite mix. In agreement with [17] who reported that the CLA assay for allergen specific IgE offers reliable results concordant with skin test. However, at the time the CLA study revealed that 65% of the studied patients are allergic to cat dander; only 20% of the same patients were allergic to cat dander by skin prick testing with a 55% concordance. The discrepancies between the two methods can be due to multiple factors, one of which is the differences in the underlying immunologic basis of the two tests. Skin testing is an in vivo biologic test that mimics the natural immediate-type hypersensitivity reaction, i.e., contact between the allergen and its specific IgE antibody on the mast cell, resulting in the local release of mediators and the formation of wheal-and-flare. On the other hand, CLA is an in vitro measurement of the level of circulating IgE antibodies in the serum, which 479

6 Fig. 1: Diagnostic indices of chemiluminescent technique for detection of sensitive allergens in children with atopic diseases in comparison to the gold standard skin prick test. The figure shows that CLA technique has a sensitivity of 97.7% and specificity of 73.9%; a positive predictive value of 70% and a negative predictive value of 98.1%. may not reflect the tissue-fixed IgE antibodies. Moreover, we observed that the remaining 18 patients who showed positive cat allergen by CLA and not by SPT, were all positive to dog allergen by CLA, this [18] cross reactivity was also stated by. Also there are differences in the allergenic quantity between the extracts used in skin testing and those used for in vitro [19] testing. This opinion was supported by who reported marked variations in the efficiency of various in vitro assays for specific serum IgE antibodies and of various skin testing techniques. In this study the overall concordance between skin [20] testing and in vitro CLA was 77.5 % while reported much higher overall concordance of 93%. Considering that the majority of the cases had bronchial asthma (23 cases; 57.5%), the CLA revealed that all our asthmatic patients are allergic to Housedust, Mite pterony and Mite farinae. These results were in accordance to other studies which demonstrated that House dust mites constitute an important indoor [21,22] allergen. Exposure to dust mite allergens is a known risk factor for sensitization and is a trigger for [23,24] asthma attacks. Dust mites are ubiquitous in most humid and warm areas and this may explain why most of our asthmatic patients showed also positivity to Aspergillus, Candida, Penicillium, Cladosporium and Alternaria. Reducing exposure to indoor allergens, especially in genetically susceptible children, can reduce the development of allergic sensitization to house dust mites and cat allergens, and this may prevent childhood asthma and decrease the frequency and severity of [25,26] asthma attacks. Identification of food allergens can be an important factor in the diagnosis of atopic dermatitis in [15] children. Although we had a little number of patients presenting with atopic dermatitis, but all of them 100% were allergic to peanut. The correlation between the total IgE and specific IgE of the different allergens was studied by Pearson correlation test where no significant correlation was [27] found. This agrees with who stated that elevated total serum IgE is a non-specific phenomenon and is of minimal value in identifying a specific allergy. 2.5% of the studied cases were negative for both total and specific IgE while they were symptomatizing and this could be explained by being allergic to other allergens not included in the panel used in this study. Other clinical studies demonstrated that some individuals have normal total IgE levels and negative values for allergen-specific IgE whilst presenting allergic symptoms. They suggested that there might be local IgE production in skin, intestinal and respiratory mucosa. Local IgE production can be detected in nasal [28] [29] [30] mucosa, bronchoalveolar lavage and stool, and their levels are not necessarily correlated to those found in blood. In conclusion the in vitro CLA assay for allergenspecific IgE may be useful as part of an initial allergy evaluation because of the high negative predictive value of negative skin test results. For the majority of allergens, the sensitivity was high however, the 480

7 specificity of in vitro tests was lower than the sensitivity, indicating that positive in vitro test results should be evaluated carefully in conjunction with clinical symptoms and allergen-specific skin tests to determine the clinical relevance of the allergen sensitization, yet chemiluminescent test is a sensitive quantification method as it fulfils many of the requirements for good evaluation being time saving, precise, with very low detection limits and large linear concentrations that can be measured with excellent regression coefficient. ACKNOWLEDGMENTS This work was supported by Department of Clinical and Chemical Pathology at National Research Centre (NRC), Department of Clinical and Chemical Pathology, Cairo University, Cairo, Egypt. We also thank Pediatric Department of the National Research Centre, Cairo University and Vacsera centre in Cairo for their cooperation with us in collecting samples. At last the whole team work would like to express their sincere gratitude and deep appreciation to late Prof.Dr. Sohier El- Bassiouny for her most generous help, advice and valuable suggestions. REFERENCES 1. Beasley, R., J. Crane, C.K. Lai, N. Pearce, Prevalence and Etiology of Asthma. J Allergy Clin Immunol. Feb., 105(2 Pt 2): S Wahn, U. and E. von Mutius, Childhood Risk Factors for Atopy and the Importance of Early Intervention. J Allergy Clin Immunol. Apr., 107(4): Nimmagadda, S.R. and R. Evans, Allergy: Etiology and epidemiology. Pediatr Rev., 20(4): 111-5, quiz; 116.reveiw. 4. Bousquet, J., P. Van Cauwenberge, N. Khaltaev, Aria Workshop Group; World Health Organization. Allergic rhinitis and its impact on asthma. J Allergy Clin Immunol. Nov., 108(5 Suppl): S Hamilton, R.G. and N.F. Adkinson, In vitro assays for the diagnosis of IgE mediated disorders. J Allergy Clin Immunol. Aug., 114(2): quiz 226. Review. 6. Vesterberg, O. and L. Holmquist, Immunochromatographic direct sampling on filter testing for aeroallergens. J. Biochem Biophys Methods, 30: Hitachi Chemical span Diagnostics, htpp/ 8. Robinson, M., J. Smart, Allergy testing and referral in children. Aust Fam Physician. Apr., 37(4): Dowdee and Osseqe, Assessment of childhood allergy for the primary care practitioner. Journal of the American academy for nurse practioners, 2: Bousquet, P.J., S. Chinn, C. Janson, M. Kogevinas, P. Burney, D. Jarvis, European Community Respiratory Health Survey; Geographical variation in the prevalence of positive skin tests to environmental aeroallergens in the European Community Respiratory Health Survey. Allergy; 62(3): Emad A. Koshak, Kamal J. Daghistani, Tarek S. Jamal, et al., Allergy Workup in Allergic Rhinitis at Jeddah, Saudi Arabia: The Internet Journal of Health, 5(1). 12. Chinoy, B., E. Yee, S.L. Bahna, Skin testing versus radioallergosorbent testing for indoor allergens. Clin Mol Allergy; Apr., 3(1): Neal, S. Jacqueline Arlian, G. Larry, Morgan, S. Marjorie, Relationship among house-dust mites, Der 1, Fel d 1,and Can f 1 on clothing and automobile seats with respect to densities in houses. Annals of Allergy, Asthma and Immunology, 88(4): (6). 14. Adedoyin, J., H. Grönlund, H. Oman, S.G. Johansson, M. van Hage, Cat IgA, representative of new carbohydrate cross-reactive allergens. J Allergy Clin Immunol; Mar., 119(3): Perry, T.T., M.K. Conover-Walker, A. Pomés, M.D. Chapman, R.A. Wood, Distribution of Peanut Allergen in the Environment. J Allergy Clin Immunol. May, 113(5): Somani, V.K., A study of allergen-specific IgE antibodies in Indian patients of atopic dermatitis. Indian J. Dermatol Venereol Leprol. Mar-Apr., 74(2): Christensen, S.N., V. Backer, L.M. DuBuske, H. Nolte, In Vitro Diagnostic Evaluation of Patients with Inhalant Allergies. Allergy and asthma Poc., 24: Kim, H.S., D.J. Kim, S.G. Lee, Analysis of Simultaneous Positivity to Multiple Allergens on MAST CLA Test. Korean J Lab Med. Dec., 25(6): Hamilton, R.G. and N.F. Jr. Adkinson, Clinical laboratory assessment of IgE-dependent hypersensitivity. J Allergy Clin Immunol. Feb., 111(2 Suppl): S Review. 20. Pfannenstiel, C., Sensitivity and Specificity Evaluation Study in Pediatric Patients: CLA Atopy Panel 20 and CAP-FEIA-System vs Skin Prick Test as "Gold Standard," Pediatrische Allergologie, Aachen, Germany. 481

8 21. El-Hefny, A., Z. Hadad and E. Ekaldious, et al., Asthma in Egyptian children: An epidemiological, environmental, clinical and immunological study. Final progress report of FRCU Grant., No Natural Heart Lung and Blood Institute and World Health Organization (NHLBI/WHO), Global Initiative for Asthma Management and prevention. Workshop, April Mihrshahi, S., J.K. Peat, G.B. Marks, C.M. Mellis, E.R. Tovey, K. Webb, W.J. Britton, S.R. Leeder, Childhood Asthma Prevention Study. Eighteen-month outcomes of house dust mite avoidance and dietary fatty acid modification in the Childhood Asthma Prevention Study (CAPS). J Allergy Clin Immunol; Jan., 111: Currie, G.P., C.M. Jackson, D.K. Lee, B.J. Lipworth, Determinants of airway hyperresponsiveness in mild asthma. Ann Allergy Asthma Immunol, 90: Peroni, D.G., A. Chatzimichail, A.L. Boner, Food allergy: what can be done to prevent progression to asthma? Ann Allergy Asthma Immunol., 89: Arshad, S.H., B. Bateman, S.M. Matthews, Primary prevention of asthma and atopy during childhood by allergen avoidance in infancy: a randomized controlled study. Thorax. Jun, 58(6): Adrian Morris, Allergen skin prick testing. Current Allergy & Clinical Immunology, 19(1). 28. Smurthwaite, L. and S.R. Durham, Local IgE synthesis in allergic rhinitis and asthma. Curr Allergy Asthma Rep.; May, 2(3): Wilson, D.R., T.G. Merrett, E.M. Varga, L. Smurthwaite, H.J. Gould, M. Kemp, et al., Increases in allergen-specific IgE in BAL after segmental allergen challenge in atopic asthmatics. Am J Respir Crit Care Med., 165(1): Sasai, K., S. Furukawa, K. Kaneko, K. Yabuta, M. Baba, Fecal IgE levels in infants at 1 month of age as indicator of atopic disease. Allergy, 49(9):