Development of a Bespoke Saliva Collector Prototype
Chapter 7: General Discussion and Future Directions
436 7.4. Patient-reported outcomes: their value in self-management protocols 440
7.5. Conclusion and Future Directions 443
References
448Appendix 1
479Appendix 2
488Figure 1.1: COPD pathogenesis 12
Figure 1.2: Combined COPD Assessment 26
Figure 1.3: Location of major and minor salivary glands 46
Figure 1.4: Saliva functions and composition 48
Figure 1.5: Lateral flow assay architecture 56
Figure 1.6: Molecular structure of C-Reactive Protein (CRP) 64
Figure 1.7: Molecular structure of Procalcitonin (PCT) 73
Figure 1.8: Molecular structure of Neutrophil Elastase (NE) 67 Figure 2.1: SalivaBio (Salimetrics, USA) passive drool collection device 82
Figure 2.2: UltraSAL-2 passive drool collection device 82
Figure 2.3: A marked 15ml centrifuge tube (Nunc, Denmark) 84
Figure 2.4: Salivary CRP ELISA kit (Salimetrics, USA) 92
Figure 2.5: Typical CRP “line-of-best-fit for a salivary CRP ELISA. 95 Figure 2.6: Salivary CRP recovery with increasing CRP concentrations 104 Figure 2.7: Salivary CRP recovery across increasing dilution 106 Figure 2.8: VIDAS B.R.A.H.M.S PCT kit (bioMérieux, France) 113 Figure 2.9: Comparison between PBS-T and Control levels 120
Figure 2.13: High range salivary PCT recovery in healthy smokers 132
Figure 2.14: Recombinant PCT levels in PBS-T 138
Figure 2.15A-B: Recombinant PCT levels in healthy non-smoker saliva 141 Figure 2.15C: Recombinant PCT levels in healthy non-smoker saliva 142
Figure 2.16A-B: FTIR Spectra 145
Figure 2.16C-D: FTIR Spectra 146
Figure 2.17: PMN Elastase ELISA kit (Immundiagnostik, Germany) 150 Figure 2.18: Typical four parameters logistic regression curve 153
Figure 2.19: Low range spiked saliva NE recovery 164
Figure 2.20: High range spiked saliva NE recovery 166
Figure 2.21: Pilot saliva sampling protocol 176
Figure 2.22: Saliva flow time in COPD patients at fixed increasing fast times 185 Figure 2.23: Salivary CRP levels in saliva at increasing fast time in COPD patients 190 Figure 2.24: Salivary PCT levels at increasing fast time in COPD patients 193 Figure 2.25: Salivary NE levels at increasing fast time in COPD subjects 196
Figure 2.26: Transferrin assay test strip 195
Figure 2.27: Urine reagent strip and colour test 197
Figure 2.28: Refined saliva sampling protocol 209
Figure 2.29: COPD wellbeing score – manual self-assessment diary 212
subjects 224 Figure 3.2: Baseline variability of salivary CRP levels across healthy non-smokers, health
smokers and stable COPD 225
Figure 3.3: Salivary PCT levels from healthy non-smokers, healthy smokers, stable COPD
subjects 227
Figure 3.4: Baseline variability of salivary PCT levels across healthy non-smokers, healthy
smokers and stable COPD 228
Figure 3.5: Salivary NE from healthy non-smokers, healthy smokers and stable COPD subjects 230 Figure 3.6: Baseline variability of salivary NE levels across healthy non-smokers, healthy
smokers and stable COPD 231
Figure 3.7A-B: Correlations of COPD patients’ PRO and salivary biomarkers levels 235 Figure 3.7C: Correlations of COPD patients’ PRO and salivary biomarkers levels 236 Figure 3.8A-B: Salivary biomarkers in COPD exacerbations 240 Figure 3.8C: Salivary biomarkers in COPD exacerbations 241 Figure 3.9A-B: Correlations between same subject serum and salivary biomarkers 243 Figure 3.9C-D: Correlations between same subject serum and salivary biomarkers 244 Figure 3.9E-F: Correlations between same subject serum and salivary biomarkers 245 Figure 4.1A: Electronic Wellbeing and Self-Assessment diary log-in screen shots 261 Figure 4.1B: Electronic Wellbeing and Self-Assessment diary log-in screen shots 262
265
Figure 4.3: Research team structured clinical log 266
Figure 4.4A: The Research team section screen shots 267
Figure 4.4B: The Research team section screen shots 268
Figure 4.5A: Host-website screenshot 286
Figure 4.5B: Host-website screenshot 287
Figure 4.6: Stable and prodromal phase COPD Wellbeing scores 290
Figure 4.7: Exacerbation phase COPD Wellbeing score 293
Figure 4.8: Cumulative Sum chart of COPD Wellbeing score 294 Figure 4.9: Post Exacerbation-Recovery COPD Wellbeing score 296 Figure 4.10: Forced expiratory volume in one second across the 4 defined COPD phases 300 Figure 4.11: Forced expiratory volume in one second clusters in the stable-prodromal period
304 Figure 4.12: Forced vital capacity across the 4 defined COPD phases 308 Figure 4.13: Forced vital capacity cluster across stable-prodromal phases 311 Figure 4.14: Salivary C-reactive protein (CRP) levels across the 4 defined COPD phases in the
whole patient exacerbator group 315
Figure 4.15: Salivary C-reactive protein (CRP) levels across the 4 defined COPD phases in the
patients who experienced raised analyte levels 316
Figure 4.16: ROC curve of salivary CRP levels 318
Figure 4.18: Salivary Procalcitonin (PCT) levels across the 4 defined COPD disease phases in
the whole exacerbator group 328
Figure 4.19: Salivary Procalcitonin (PCT) levels across the 4 defined COPD phases in patients
who experienced a raised analyte levels 329
Figure 4.20: ROC curve of salivary PCT levels 331
Figure 4.21: Salivary PCT clusters in the stable-prodromal phases 335 Figure 4.22: Salivary Neutrophil Elastase (NE) levels across the 4 defined COPD phases in the
whole exacerbator cohort 339
Figure 4.23: Salivary Neutrophil Elastase (NE) levels across the 4 defined COPD phases in
patients who experienced raised analyte levels 340
Figure 4.24: Salivary NE clusters across the stable-prodromal phases 344 Figure 4.25: Factor plot of the 3 exacerbation phenotypes 353
Figure 5.1: Populations and workshop recruitment 368
Figure 6.1: Workshop recruitment 387
Figure 6.2: Ergonomic lip feature 395
Figure 6.3: First (conic) Filter 397
Figure 6.4: Cellulose acetate filter 398
Figure 6.5: 3D-printed prototype saliva collector 400
Figure 6.9: Salivary CRP levels across different saliva processing methods 413 Figure 6.10: Salivary PCT levels across different saliva processing methods 415 Figure 6.11: Salivary NE levels across different saliva processing levels 417 Figure 6.12: Unadulterated compared to collector filtered standard NE levels 421
Table 1.1: 2001 GOLD classification of COPD severity 23 Table 1.2: Updated COPD severity classification GOLD 2003 24
Table 1.3: Modified MRC dyspnoea score 28
Table 1.4: Summary of COPD scoring systems 30
Table 1.5: Technological approaches to salivary analysis 62
Table 2.1: Thesis subjects’ demographics and allocation 89
Table 2.2: Generic salivary CRP ELISA plate layout 94
Table 2.3: COPD patient demographics 97
Table 2.4: Saliva spiking experimental protocol 99
Table 2.5: Saliva dilution experimental protocol 99
Table 2.6: The dilution and recovery experiment protocol for each COPD patients’ sample of
saliva 100
Table 2.7: Salivary CRP levels and recovery across increasing spiked concentrations 103 Table 2.8: Endogenous salivary CRP levels and recovery percentage at increasing dilutions
107 Table 2.9: Manufacturer endogenous salivary CRP levels and recovery percentage at increasing
dilutions 108
Table 2.13: High range PCT spiking experimental protocol 118 Table 2.14: Endogenous PCT levels in saliva from healthy individuals 121 Table 2.15: Low range PCT levels and recovery in healthy non-smokers 123 Table 2.16: High range PCT levels and recovery in healthy non-smokers 126 Table 2.17: Low range PCT levels and recovery in healthy smokers 129 Table 2.18: High range PCT levels and recovery in healthy smokers 131 Table 2.19A: Protocol for PBS-T spiked with recombinant PCT experiment 135 Table 2.19B: Protocol for recombinant PCT neat saliva spike experiment 136
Table 2.20: Recovery of PCT in spiked PBS-T samples 137
Table 2.21: Recombinant PCT recovery in spiked saliva 140
Table 2.22: Generic NE microtitre plate layout 151
Table 2.23: Group demographics 156
Table 2.24: Dilution saliva NE experiment microtitre plate layout 157 Table 2.25: Spiked saliva NE experiment microtitre plate layout 157 Table 2.26: Unadulterated saliva dilution experimental protocol 158 Table 2.27: Low range NE spiking experimental protocol 159 Table 2.28: High range NE spiking experimental protocol 160 Table 2.29: Endogenous salivary NE levels across varying dilutions in all subjects 162
subjects (n = 5) 163 Table 2.31: High range salivary NE levels across varying dilutions in both COPD and healthy
subjects (n = 5) 165
Table 2.32: Demographic details for participating healthy and COPD patients 180 Table 2.33: A comparison of the time taken by all healthy subjects and COPD patients to produce a set volume of saliva (2mls), with associated saliva flow rates 182 Table: 2.34: A comparison of the time taken to produce a set volume of saliva (2mls) at different fast times after eating and associated saliva flow rates in COPD patients 183 Table 2.35: Saliva CRP levels at increasing fast time in COPD patients 189 Table 2.36: Saliva CRP significance levels at increasing fast time in COPD patients 189 Table 2.37: Saliva PCT levels at increasing fast time in all COPD patients 192 Table 2.38: Salivary NE levels at increasing fast time in all COPD patients 194
Table 2.39: Demographic details of COPD patients 204
Table 2.40: Demographic breakdown of all saliva samples tested for blood contamination 206 Table 3.1: Subject demographics and salivary biomarker profiles for the healthy non-smokers,
smokers and stable COPD subjects (n = 107) 222
Table 3.2: Correlations of whole study population (n = 143) symptom scores and sputum
salivary biomarker levels 234 Table 3.4: Same COPD patients in stable and exacerbation phase (n = 36) demographics,
salivary biomarker levels and symptom profiles 238
Table 4.1: Data classification definitions 275
Table 4.2: Total study population baseline and stable phase characteristics 280 Table 4.3: Exacerbation and non-exacerbators cohort characteristics 281 Table 4.4: Total length of pre-exacerbation phase in the exacerbation cohort 282 Table 4.5: Single and re-exacerbator cohort characteristics 284 Table 4.6: COPD Wellbeing Score sub-populations based on stable-prodromal data 291 Table 4.7: Comparison of COPD Wellbeing Scores between single exacerbator and
re-exacerbator patients 297
Table 4.8: FEV1 values across the whole COPD study cohort 301 Table 4.9: LCGA Goodness of fit tests for stable-prodromal FEV1 data 302 Table 4.10: Homogeneity of COPD patient cluster membership for stable-prodromal FEV1 data 302 Table 4.11: Exacerbator COPD patient cluster membership for stable-prodromal FEV1 data
303 Table 4.12: Covariates between the 2 patient exacerbator clusters for FEV1 and control
non-exacerbator cohort 306
Table 4.13: FVC values across the whole study cohort 309
Table 4.15: Spirometry values between single and re-exacerbators 312 Table 4.16: Salivary CRP levels across the whole COPD study cohort 314 Table 4.17: Sensitivity and specificity of exacerbation phase salivary CRP levels 317 Table 4.18: LCGA Goodness of fit tests for stable-prodromal salivary CRP levels 320 Table 4.19: Homogeneity of COPD patients cluster membership for stable-prodromal salivary
CRP levels 320
Table 4.20: Exacerbator COPD patient cluster membership for stable-prodromal salivary CRP
levels 321
Table 4.21: Multinomial regression of covariates between the 3 exacerbator sub-population
clusters for salivary CRP 325
Table 4.22: Covariates comparisons between the 3 patient exacerbator clusters and
non-exacerbator control cohort for salivary CRP 326
Table 4.23: Salivary PCT levels across the whole COPD study cohort 327 Table 4.24: Sensitivity and specificity of exacerbation phase salivary PCT levels 330 Table 4.25: LCGA Goodness of fit tests for stable-prodromal salivary PCT levels 333 Table 4.26: Homogeneity of COPD patients cluster membership for stable-prodromal salivary
PCT levels 333
Table 4.27: COPD patients cluster membership for stable-prodromal salivary PCT levels 334
Table 4.30: LCGA Goodness of fit tests for stable-prodromal salivary NE levels 342 Table 4.31: Homogeneity of COPD patients cluster membership for stable-prodromal salivary
NE levels 342
Table 4.32: COPD patients cluster membership for stable-prodromal salivary NE levels 343 Table 4.33: Multinomial regression of covariates between the 3 exacerbator sub-population
clusters for salivary NE 346
Table 4.34: Covariate comparison between the 3 patient exacerbator clusters for salivary NE
and the non-exacerbator control cohort 347
Table 4.35: Comparison of salivary biomarker levels between single and re-exacerbator patient
cohorts 349
Table 4.36: Multidimensional composite score for each COPD exacerbator patient 351
Table 5.1: Workshop demographics 369
Table 5.2: Workshop questions 371
Table 6.1: Workshop demographics 388
Table 6.2: Workshop questions 389
Table 6.3: Subject demographics 407
Table 6.4: Subject demographics 411
Table 6.5: Endogenous salivary CRP levels: Tube centrifugation compared to modified saliva
collector in the same samples 412
collector in all healthy subjects 414 Table 6.7: Endogenous salivary NE levels: Tube centrifugation compared to modified saliva
collector in the same healthy subjects 416
Table 6.8: Recovery of “standard” NE using two saliva sample processing methods 420
Dedicated to Jaimini Patel (Mum)
“alma mater”