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Aerobiology, Image Analysis and Allergenicity of Pollen

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Aerobiology, Image Analysis and Allergenicity of Pollen AEROBIOLOGY, IMAGE ANALYSIS AND ALLERGENICITY OF POLLEN AND SPORES IN SINGAPORE ONG TAN CHING NATIONAL UNIVERSITY OF SINGAPORE 2004 AEROBIOLOGY, IMAGE ANALYSIS AND ALLERGENICITY OF ONG TAN CHING 2004 POLLEN AND SPORES IN SINGAPORE AEROBIOLOGY, IMAGE ANALYSIS AND ALLERGENICITY OF POLLEN AND SPORES IN SINGAPORE ONG TAN CHING B. Sc. (Honours), Universiti Putra Malaysia A THESIS SUBMITTED FOR THE DEGREE OF DOCTOR OF PHILOSOPHY DEPARTMENT OF PAEDIATRICS NATIONAL UNIVERSITY OF SINGAPORE 2004 Acknowledgements I would like to thank my supervisors, Adjunct Associate Professor Lee Bee Wah, Associate Professor Hugh Tan Tiang Wah and Dr. Chew Fook Tim for their immense support, guidance and patience during the course of my study and the opportunity to undertake this project not forgetting Associate Professor Tan Teck Koon with his valuable advice on mycology based work. I would like to thank Wang Xiaoshan for being my mentor in statistics and Wong Fei Ling for teaching me the ropes in mycological research and Dr. Adrian Loo, Dr. Bi Xuezhi and Dr. Shang Huishen for the guidance and helpful problem solving suggestions. I am also very grateful to Ong Seow Theng, Tan Teng Nging, Wang Wun Long, Hon Sook Mei, Kuay Kuee Theng and Hao Jing for their relentless encouragement and the laughter and pain shared during the process of pursuing their graduate studies. I would also like to extend my gratitude to my colleagues especially Lim Puay Ann and those working in the Functional Genomics Laboratory 1 and 3 for the wonderful experience working together and their never failing encouragement and support. I would also like to specially thank T. Morgany for helping me out with the airspora traps, sample collections and preparation for the image analysis. Finally, my thanks to my family who provided me with unconditional love and support to undertake this challenge and complete it to what it is today. i Table of Contents Page Acknowledgements i Table of contents ii List of figures ix List of tables xii List of abbreviations xiv Summary xvi CHAPTER 1: INTRODUCTION 1.1 ALLERGY 1 1.1.1 Hypersensitivity 1 1.1.1.1 Allergy ─ Type I hypersensitivity 1 1.2 ALLERGENS 3 1.2.1 Fungal allergenicity 3 1.2.2 Pollen allergenicity 6 1.2.2.1 Tree pollen allergenicity 7 1.2.2.2 Dicotyledonous weed pollen allergenicity 8 1.2.2.3 Grass pollen allergenicity 9 1.2.3 Fern allergenicity 11 1.3 TRENDS IN ALLERGIC DISEASES 12 ii CHAPTER 2: AEROBIOLOGY IN SINGAPORE 2.1 INTRODUCTION 2.1.1 Singapore 14 2.1.2 Airspora 14 2.1.2.1 Fungal spores 15 2.1.2.2 Fern spores 16 2.1.2.3 Pollen 16 2.1.3 Technical factors influencing aispora quantification 16 2.1.4 Effects of airspora counts on health 17 2.1.5 Aims 18 2.2 MATERIALS AND METHODS 2.2.1 Airspora sampling and meteorological data 19 2.2.2 Evaluation and optimisation of screening factors 20 2.2.3 Seasonal patterns 22 2.2.4 Diurnal patterns 23 2.2.5 Statistical analyses 23 2.3 RESULTS 2.3.1 Evaluation and optimisation of screening factors 24 2.3.1.1 Screening magnifications 24 2.3.1.2 Position of traverses 25 2.3.1.3 Number of traverses 25 2.3.1.4 Orientation of traverses 26 iii 2.3.2 Seasonal patterns 26 2.3.2.1 Fungal spores 26 2.3.2.2 Unidentified fungal spore 34 2.3.2.3 Pollen 36 2.3.2.4 Fern spores 36 2.3.2.5 Comparisons of counts between different stations 37 2.3.2.6 Association with meteorological variables 38 2.3.3 Diurnal patterns 47 2.3.3.1 Fungal spores 47 2.3.3.2 Pollen 50 2.3.3.3 Fern spores 53 2.3.3.4 Association with meteorological parameters 53 2.4 DISCUSSION 2.4.1 Evaluation and optimisation of screening factors 58 2.4.2 Seasonal and diurnal patterns 60 2.4.2.1 Fungal spores 60 2.4.2.2 Pollen 64 2.4.2.3 Fern spores 67 2.4.3 Comparison of counts between different stations 68 2.5 CONCLUSIONS 69 iv CHAPTER 3: IDENTIFICATION OF AIRSPORA COMPONENTS BY IMAGE ANALYSIS 3.1 INTRODUCTION 3.1.1 Shortcomings of current airspora quantification methods 70 3.1.2 Pollen grain identification 72 3.1.3 Fern spore identification 72 3.1.4 Fungal spore identification 73 3.1.5 Aims 73 3.2 MATERIALS AND METHODS 3.2.1 Samples preparation 74 3.2.2 Image capture 77 3.2.3 Features measurement 77 3.2.4 Statistical analyses 78 3.3 RESULTS 3.3.1 Local airspora 80 3.3.2 Grass (Poaceae) pollen 86 3.3.3 Asteraceae weed pollen types 92 3.3.4 Olea look-alike pollen types 98 3.3.5 All pollen types 104 3.3.6 Cluster analyses 110 3.4 DISCUSSION 115 3.5 CONCLUSIONS 119 v CHAPTER 4: DEVELOPMENT OF A DOT IMMUNOARRAY SYSTEM FOR SIMULTANEOUS DETECTION OF A LARGE ARRAY OF ALLERGEN-SPECIFIC IGE MOLECULES 4.1 INTRODUCTION 4.1.1 Techniques in allergy diagnosis 125 4.1.2 Measurement of IgE levels 126 4.1.3 Advantages of in vitro techniques 127 4.1.4 Limitations of the available techniques and aims of the 128 study 4.1.5 Aims 128 4.2 MATERIALS AND METHODS 4.2.1 Dotting apparatus 129 4.2.2 Support materials and washing buffers 129 4.2.3 Loading efficiency of the 384-pin MULTI-BLOT™ 130 replicator 4.2.4 Allergen extracts 130 4.2.5 Allergen array for the detection of specific IgE 131 4.2.6 Patients and sera 135 4.2.7 Allergen array validation 135 vi 4.2.8 Statistical analyses 137 4.3 RESULTS 4.3.1 Support materials, washing buffer and loading efficiency 139 4.3.2 Performance of allergen array 142 4.3.3 Allergen array validation 149 4.3.3.1 Immunoarray versus ELISA 149 4.3.3.2 Immunoarray versus UniCAP® 149 4.4 DISCUSSION 153 4.5 CONCLUSIONS 156 CHAPTER 5: AIRSPORA ALLERGY IN SINGAPORE 5.1 INTRODUCTION 5.1.1 Prevalence of airspora allergies 158 5.1.2 Aims 160 5.2 MATERIALS AND METHODS 160 5.3 RESULTS 5.3.1 Detected frequencies of specific IgEs to airspora allergens 161 5.3.2 Cluster analyses 167 5.4 DISCUSSION 175 5.5 CONCLUSIONS 190 vii CHAPTER 6: SIGNIFICANCES, SUMMARY 191 AND FUTURE RESEARCH WORK REFERENCES 195 APPENDICES i - xxx viii List of figures Page Figure 1.1 Proposed cellular and molecular mechanism of allergy. 2 Adapted from Holgate (1999). Figure 2.1 Locations of the sampling and meteorological stations. 20 Figure 2.2 Slide screening methods used: (A) five horizontal 21 traverses (3 mm apart) and (B) 12 vertical traverses (4 mm apart). Orientation of traverses: Longitudinal = L and transverse = T. Figure 2.3 Scatter plots and comparisons of counts made at 250× and 27 400× magnifications for a) all airspora types, b) airspora <200µm2 in size and c) airspora >200µm2 in size using Spearman’s Correlation Test (Correlation coefficient = r) and Wilcoxon Rank Test. p-value: p<0.001***, p<0.01**, p<0.05* . Figure 2.4 Examples of fungal spore counts <200 µm2 in area 28 screened at 250× and 400× magnifications. Figure 2.5 Comparisons of airspora counts at different horizontally 29 positioned traverses (H1 to H5) along the length of the slide using the Wilcoxon Rank Test. a) all airspora types, b) airspora <200µm2 and c) airspora >200µm2. p-value: p<0.001***, p<0.01** and p<0.05*. Figure 2.6 Scatter plots and count comparisons for different numbers 30 of screening traverses for all airspora types using Spearman’s Correlation Test (correlation coefficient = r) and Wilcoxon Rank Test. p-value: p<0.001***, p<0.01** and p<0.05*. Figure 2.7 Scatter plots and count comparisons using different 31 numbers of screening traverses for airspora <200 µm2 in area size using Spearman’s Correlation Test (correlation coefficient = r) and the Wilcoxon Rank Test. p-value: p<0.001***, p<0.01** and p<0.05*. Figure 2.8 Scatter plots and count comparisons using different 32 numbers of screening traverses for airspora >200µm2 in area size using the Spearman’s Correlation Test (correlation coefficient = r) and the Wilcoxon Rank Test. p-value: p<0.001***; p<0.01** and p<0.05*. Figure 2.9 Scatter plots and count comparisons from horizontal and 33 vertical traverses using the Spearman’s Correlation Test (correlation coefficient = r) and the Wilcoxon Rank Test. p-value: p<0.001***, p<0.01** and p<0.05*. Figure 2.10 Seasonal patterns of major fungal spores from the Kent 41 3 Ridge Station. Fungal spore counts are in number of fungal spores m -1 day . ix List of figures Page Figure 2.11 Photomicrographs of unknown spore “kuaci” 35 Figure 2.12 Seasonal patterns of the ascospore ‘kuaci’ from the Kent 42 Ridge Station. Figure 2.13 Seasonal patterns of pollen types and airspora from the 43 Kent Ridge Station. Pollen counts are in number of pollen grains m3 day- 1. Figure 2.15 Seasonal patterns of fern spores from 1991 to 1995 at the 44 Kent Ridge Station. Fern spore counts are in number of fern spores m-3 day-1. Figure 2.16 Diurnal calendars for Cladosporium spp., Didymosphaeria 48 sp. and the ascospore ‘kuaci’ for 1995, 1996, 1997 and average of all 3 years.
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