Tables
- Table 1– Clinimetric properties of biomarkers
Definition Justification of importance Reliability Degree to which a measurement is consistent and free from error Important to quantify error (systematic and random) so that true changes can be discerned from changes due to normal fluctuations Validity The gold standard outcome measures are often not feasible; therefore, it is important to know how an alternative outcome measure compares to the gold standard, and how different outcome measures compare It is important to know the ability of outcome measures to discriminate between different groups Concurrent validity Degree to which a test correlates with a “gold standard” criterion test which has been established as a valid test of the attribute of interest Convergent validity Degree to which a test correlates with another test which measures the same attribute Discriminate validity Degree to which a test differentiates between groups of individuals known to differ in the attribute of interest Predictive validity Degree to which an attribute can be predicted using the result of a predictor test/or degree to which prognosis can be predicted Responsiveness Degree to which a test changes in response to an intervention known to alter the attribute of interest Important attribute of tests used in clinical practice or research to assess treatment benefit (e.g. to identify improvements in response to an intervention) Reproduced from [1] with permission.
- Table 2– Discriminate validity of bronchoalveolar lavage (BAL) inflammatory markers between cystic fibrosis (CF) patients and non-CF subjects
First author [ref.] Aliquot fraction analysed CF Non-CF PMN cell counts ×10 3 cells·mL−1, unless otherwise stated PMN cells % Neutrophil elastase IL-6 ×103·mL−1, unless otherwise stated IL-8 ×103·mL−1, unless otherwise stated Statistical tests Data presentation Subjects n Age Subjects n Age Armstrong [26] F 50 infected 19.1 (1.5–71) months 19 12.4±11.8 months CF 50 (39–65) versus non-CF 8.2 (4.6–15)*** CF 80 (66–90) versus non-CF 32 (13–57)*** CF 1153 (754–1762) versus non-CF 24 (12–47)*** CF 50 (39–65) versus non-CF 8.2 (4.6–15)*** Wald test Geometric mean (95% CI) Armstrong [26] F 28 Infants (age not reported) 10 Infants (age not reported) CF 11 (8.7–13) versus non-CF 2.1 (0.95–4.7) μg·mL−1*** Wald test Geometric mean (95% CI) Muhlebach [38] Not specified 17 25±4 months 25 15±3 months CF 41.2±7.0 versus non-CF 21.8±5.2* CF 6868±2065 pg·mL−1 versus non-CF 1434±362 pg·mL−1¶ CF 41.2±7.0 versus non-CF 21.8±5.2* Mann–Whitney U-test Mean±sem Armstrong [39] F 5 virus infected Infants
(age not reported)13 Infants
(age not reported)CF 169 (16–1809) versus non-CF 7 (2–22)*** CF 1372 (561–3358) versus non-CF 32 (12–84)*** ANCOVA Geometric mean (95% CI) Khan [31] P 16 0.48 (0.08–0.97) years 11 1.02 (0.25–2.7) years CF 673.6±367.9 versus non-CF 6.9±1.7+ CF 1.18±0.46 μg·mL−1 versus non-CF 0 μg·mL−1§ CF 1298±338 pg·mL−1 versus non-CF 92±25 pg·mL−1*** Independent t-test Mean±sem Kirchner [40] P 16 10.5 (2–25) months 9 (disease) 11.7 (3–30) months ¶ Kruskal–Wallis Values not reported Muhlebach [41]#,¶ Not specified 51 1.8 years (3 weeks–13 years) 50 (disease) 1.0 years (2 weeks–8.3 years) *** * *** Independent t-test Values not reported Muhlebach [42]# Not specified 55 3.7 (0.1–13) years 56 2.1 (0.04–9) years * * Independent t-test Values not reported Noah [43] P 15 (infected) 2.4 (0.1–13) years 12 (uninfected) 1.4 (0.1–8.8) years CF 3358 (100–11 520) versus non-CF 10 (1–117)* CF 84 (16–96) versus non-CF 5 (1–51)* CF 56 (17–681) versus non-CF 10 (2–59)* CF 11 614 (1424–71 100) versus non-CF 459 (21–2040)* Kruskal–Wallis Median (range) Noah [43] P 15 (infected) 2.4 (0.1–13) years 12 (infected) 1.4 (0.1–8.8) years CF 3358 (100–11 520) versus non-CF 229 (1–2976)* CF 84 (16–96) versus non-CF 46 (1–93)* ns CF 11 614 (1424–71 100) versus non-CF 1860 (18–6224)* Kruskal–Wallis Median (range) MacGregor [44] P 39 6.4±0.7 years 38 6.1±0.6 years ns CF 1308.1±56.4 versus non-CF 716±89.9*** Mann–Whitney U-test Mean±sem Hilliard [45] P 43 6.2 (0.3–16.8) years 7 (PCD) 9.2 (5.7–14.8) years ns ns ns Nonparametric tests Hilliard [45] P 43 6.2 (0.3–16.8) years 26 (CRD) 4.8 (0.9–15.2) years CF 0.7 (0.02–23.97)×106·mL−1 versus non-CF 0 (0–17.91)×106·mL−1 *** CF 201 (100–5000) μm·mL−1 versus non-CF 100 (100–1297) μm·mL−1 *** CF 1340 (31–1760) pg·mL−1 versus 703 (31–1560) pg·mL−1 *** Nonparametric tests Median (range) Hilliard [45] P 43 6.2 (0.3–16.8) years 7 (disease control) 5.8 (0.3–16.3) years CF 0.7 (0.02–23.97)×106·mL−1 versus non-CF 0 (0–0.01)×106·mL−1 *** CF 201 (100–5000) μm·mL−1 versus non-CF 100 (100–100) μm·mL−1 *** CF 1340 (31–1760) pg·mL−1 versus 64 (31–221) pg·mL−1** Nonparametric tests Median (range) Harris [46] F 29 (during exacerbation) 9.1±12 years 12 6.0±1.4 years CF 6.1±0.12 log10 versus 4.9±0.17 log10*** CF 68.8±3.6 versus 16.4±3.8*** Wilcoxon rank sum test Mean±sem Dean [37] svBAL, F 5 8 (4–18) years 8 (asthma) Adults (age not reported) CF 6618 (1200–11 136) pM versus non-CF 302.4 (48–984) pM* Not reported Mean (range) Dean [37] svBAL, F 5 8 (4–18) years 6 (PCD) Children (age not reported) CF 6618 (1200–11 136) pM versus non-CF 302.4 (48–984) pM*ƒ Not reported Mean (range) Reeves [47] Not specified 15 Children and adolescents 12 8.21±1.22 years Mann–Whitney U-test Values not reported Ratjen [48] F 105 5–37 years 48 3–15 years First aliquot
CF 52±30 versus non-CF 4±5##Mann–Whitney U-test Mean±sd Ratjen [48] P (aliquots 2 and 3) 105 5–37 years 48 3–15 years Pooled CF 29±23 versus non-CF 2±3##¶ Mann–Whitney U-test Mean±sd Reeves [49] F 18 19.6±5.2 years 14 (other disease) 50.14±10.5 years CF 325±81.96 pg·mg−1 versus 125.92±43.95 pg·mg−1 * Independent t-test Mean±sem McGarvey [6] F (cytology), P (remaining) 11 22.7±3.8 years 9 (mild asthma) 24.1±3.9 years ns ns ANOVA 11 (healthy) 24.3±2.8 years Bonfield [33] P 22 20±2 years 13 29±6 years CF 41±10×106·mL−1 versus non-CF 0.09±0.02×106·mL−1 * CF 60±5 versus non-CF 1±0.3* CF 1.9±0.5 μM versus non-CF 0 μM* p=0.06
CF 287±104 versus non-CF 0CF 32 565±9686 versus non-CF 0* Kruskal–Wallis Mean±sem Konstan [50] P 18 20±1 years 23 25±1 years CF 38±14 (1–222)×106·mL−1 versus non-CF 0.1±0.04 (0–0.6)×106·mL−1 * Independent t-test Mean±sem (range) Meyer [35] P 14 (exacerbation) 17–40 years 8 19–24 years CF 51.1±5.2 versus non-CF 0.3±0.4* CF 323±127 nM·min−1·mL−1 versus non-CF 0 nM·min−1·mL−1* Independent t-test Mean±sem Nakamura [51] Not specified 8 28±2 years 7 29±3 years CF 101±26 versus non-CF 0.4±0.3* CF 31±10 versus non-CF 0* CF 35±13 versus non-CF 0* Not reported Mean (range) All four biomarkers are present at higher levels in patients with CF. All samples are BAL, unless otherwise stated. PMN: polymorphonuclear; IL: interleukin; F: first; P: pooled; svBAL: small volume BAL; ns: not significant; PCD: primary ciliary dyskinesia; CRD: chronic respiratory disease. #: [42] includes patients previously reported in [41]; *: p<0.05; **: p<0.01; ***: p<0.001; ¶: p<0.005; +: p=0.002; §: p=0.03; ƒ: p=0.02; ##: p<0.0001.
- Table 3– Discriminate validity of bronchoalveolar lavage (BAL) inflammatory markers between groups of patients with cystic fibrosis (CF) with different phenotypes
First author [ref.] Aliquot fraction analysed Group 1 Group 2 PMN cell counts ×103 cells·mL−1, unless otherwise stated PMN cells % Neutrophil elastase IL-6 ×103·mL−1, unless otherwise stated IL-8 ×103·mL−1, unless otherwise stated Statistical tests Data presentation Subjects n Age Subjects n Age Sly [9] F (microbiology), P (aliquots 2 and 3) 48 asymptomatic Infants 9 symptomatic Infants ns Group 1 14.5 (9.0–27.1) versus Group 2 39.7 (28.3–65.0)# Group 1 100 (100–143) ng·mL−1 versus Group 2 100 (100–2025) ng·mL−1# ns Independent t-tests Median (IQR) Sly [9] F (microbiology), P (aliquots 2 and 3) 45 uninfected Infants 12 infected Infants Group 1 31.3 (11.3–76.3) versus Group 2 169 (72.2–208.5)# Group 1 13.5 (8.3–26.1) versus Group 2 35.7 (25.3–44.7)¶ Group 1 100 (100–100) ng·mL−1 versus Group 2 350 (100–1330) ng·mL−1+ Group 1 260 (128–630) pg·mL−1 versus Group 2 770 (420–2120) pg·mL−1§ Independent t-tests Median (IQR) Armstrong [13] svBAL, P 18 infected 2.6±1.1 months 10 noninfected, nonsymptomatic 2.6±1.1 months Group 1 153 (52–449) versus Group 2 15 (8–29)*** Group 1 78 (52–94)% versus Group 2 0 (0–31)%*** Group 1 1112 (582–2126) versus Group 2 24 (9–63)*** Independent t-test Mean (95% CI) Sly [9] F (microbiology), P (aliquots 2 and 3) 24 no antistaphylococcal prophylaxis 3.6 (3.4–4.8) months 33
antistaphylococcal prophylaxis3.6 (2.8–4.7) months ns ns ns ns Independent t-tests Armstrong [26] F 28 infected Infants (age not reported) 7 pristine
17 uninfectedInfants (age not reported) Group 1 11 (8.7–13) pg·mL−1 versus Group 2 pristine 2.2 (0.48–10) pg·mL−1 and uninfected 2.2 (1.8–2.6) pg·mL−1
***Wald test Geometric mean (95% CI) Armstrong [26] F 50 infected 13 pristine Group 1 562 (407–777) versus Group 2 pristine 127 (76–210) and uninfected 106 (67–168)*** Group 1 50 (39–65) versus Group 2 pristine 8.4 (5.2–14) and uninfected 7.1 (4.4–12)*** Group 1 80 (66–90)% versus Group 2 pristine 0 (0–25)% and uninfected 20 (6–44)% of patients with free NE activity*** Group 1 1153 (754–1762) pg·mL−1 versus Group 2 pristine 32 (15–72) pg·mL−1 and uninfected 49 (23–101) pg·mL−1*** Wald test Geometric mean (95% CI) Rosenfeld [52] Not specified 31 (≥105 CFU·mL−1) ≤15 months at study entry 29 (<105 CFU·mL−1) ≤15 months at study entry ns ns Generalised estimating equation Rosenfeld [52] Not specified 31 (≥105 CFU·mL−1) ≤15 months at study entry 30 no pathogen ≤15 months at study entry Group 1 1145 (580–2264) versus Group 2 114 (63–206)*** Group 1 1802 (1104–2942) versus Group 2 427 (269–679)ƒ¶ Generalised estimating equation Geometric mean (95% CI) Rosenfeld [52] Not specified Not specified P. aeruginosa positive ≤15 months at study entry Not specified other pathogen ≤15 months at study entry ns Generalised estimating equation Nixon [53] F 20 symptomatic 20.7±10.6 months 34 asymptomatic 15.7±9.7 months ns ns ns Generalised estimating equation Nixon [53] F 15 infected 22.8±6.7 months 39 uninfected 15.9±10.9 months Group 1 59 (47–70) versus Group 2 28 (21–35) *** ns Group 1 641 (305–1350) versus Group 2 253 (158–406)##¶ Generalised estimating equation Mean and geometric mean (95% CI) Dakin [54] F 8 infected 23 months 14 uninfected 23 months 90.8% >105 pathogens versus 24.1% <105 pathogens¶¶ 4600 pg·mL−1 >105 pathogens versus 1627 pg·mL−1 <105 pathogens++ Independent t-test/ANOVA Brennan [30] F (microbiology), P (aliquots 2 and 3) 9 infected 1.55 (1.11–2.51) years 15 uninfected 1.58 (1.12–2.91) years 667 (58–5342) infected versus 60 (39–91) noninfectedƒ 52 (38–81)% infected versus 17.3 (9–29)% noninfected
***ns Not reported Median (IQR) Gutierrez [55] F 6 (≥105 CFU·mL−1) 35.3±21.3 months 24 (<105 CFU·mL−1) 22.1±13.3 months *** (right middle lobe)
ns (lingula)§§ (right middle lobe)
¶¶ (lingula)Independent t-test Armstrong [39] F 5 virus-infected 6.5±5.3 months 22 noninfected 2.6±1.5 months 169 (16, 1809) virus infected versus 16 (8, 34) noninfected*** 1372 (561, 3358) virus-infected versus 47 (21, 108) noninfected*** ANCOVA Geometric mean (95% CI) Brennan [30] F (microbiology), P (aliquots 2 and 3) 14 asymptomatic <6 years 11 symptomatic <6 years Group 1 60.3 (38.45–90.55) versus Group 2 667.58 (64.3–5341.48)ƒƒ,¶ Group 1 16.5 (10.5–26.5) versus Group 2 53 (49.67–80.5)*** ns Not reported Median (IQR) Sagel [56] P 59 P. aeruginosa positive 6 months–6 years 31 no pathogens 6 months–6 years Group 1 5.6±0.7 log10·mL−1 versus Group 2 4.8±0.6 log10·mL−1*** Group 1 49.3±25.9 versus Group 2 22.1±14.4*** Group 1 41% of patients versus Group 2 0% of patients with NE detected in BAL*** ns Group 1 3.1±0.6 pg·mL−1 versus Group 2 2.3±0.6 pg·mL−1*** Linear regression Mean±sd Sagel [56] P 21 P. aeruginosa negative, other pathogen present 6 months–6 years 31 no pathogens 6 months–6 years Group 1 59±0.3 log10·mL−1 versus Group 2 5.6±0.3 log10·mL−1*** Group 1 33.6±17.6 versus Group 2 22.1±14.4* ns ns Group 1 2.8±0.7 pg·mL−1 versus Group 2 2.3±0.6 pg·mL−1** Linear regression Mean±sd Sagel [56] P 59 P. aeruginosa positive 6 months–6 years 21 P. aeruginosa negative, other pathogen present 6 months–6 years ns Group 1 33.6±17.6 versus Group 2 22.1±14.4+ ns ns ns Linear regression Mean±sd Sagel [57] P 44 nonmucoid P. aeruginosa 6 months–6 years 15 mucoid P. aeruginosa 6 months–6 years Group 1 5.9±0.6 log10·mL−1 versus 5.5±0.7 log10·mL−1# Group 1 62.7±22.1 versus Group 2 44.5±25.7### ns ns ns Linear regression Mean±sd Sagel [56] P Not specified S. aureus present 6 months–6 years Not specified S. aureus not present 6 months–6 years *** ¶¶¶ + *** Linear regression Values not reported Hilliard [45] P 31 exacerbation 6.2 years 12 clinically well 6.2 years Group 1 1.0×106·mL−1 versus Group 2 0.4×106·mL−1* Nonparametric tests Median Muhlebach [41] Not specified Not specified P. aeruginosa positive Mean 7.6 years Not specified other pathogen Mean 1.9 years ns ns Independent t-test Armstrong [15] Not specified 14 infected Children (age not reported) 20 noninfected Children (age not reported) Group 1 259 (70–968)×106·L−1 versus 17 (8–36)×106·L−1*** Group 1 2013 (1164–3784) ng·L−1 versus Group 2 26 (13–51) ng·L−1*** Independent t-test Mean (95% CI) Noah [43] P 15 infected <13 years 10 uninfected <13 years Group 1 3358 (100–11 520) versus Group 2 291 (25–952)* Group 1 84 (16–96) versus Group 2 40 (4–62)* ns ns Kruskal–Wallis Median (range) Regamey [28] P 36 during exacerbation 10 stable Group 1 970 (287–1584) versus Group 2 222 (21–380)¶¶¶ Independent t-test Median (IQR) Regamey [28] P 13 Aspergillus present 13 Aspergillus not present ns Independent t-test Regamey [28] P 11 P. aeruginosa positive 15 P. aeruginosa negative ns Independent t-test Bonfeld [33] P 17 P. aeruginosa positive Adolescents and adults (age not reported) 5 P. aeruginosa negative Adolescents and adults (age not reported) ns Kruskal–Wallis Kirchner [40] P 7 older patients Mean (range) 18.6 (13.5–31) years 16 younger patients Mean (range) 10.5 (2–25) months ns Kruskal–Wallis Biomarkers are present at higher levels in patients with more severe lung disease. All samples are BAL, unless otherwise stated. PMN: polymorphonuclear; IL: interleukin; F: first; P: pooled; svBAL: small volume BAL; IQR: interquartile range; ns: not significant; P. aeruginosa: Pseudomonas aeruginosa; S. aureus: Staphylococcus aureus; NE: neutrophil elastase. *: p<0.05; **: p<0.01; ***: p<0.001; #: p=0.0001; ¶: p=0.027; +: p=0.003; §: p=0.036; ƒ: p=0.04; ##: p=0.03; ¶¶: p=0.01; ++: p=0.0001; §§: p=0.05; ƒƒ: p= 0.002; ###: p=0.02; ¶¶¶: p=0.004.
- Table 4– Discriminate validity of bronchoalveolar lavage (BAL) inflammatory markers
First author [ref.] Aliquot fraction analysed Subjects Age Participants Results Statistics Khan [31] P 16 0.08–0.97 years Neutrophil count
PMN cell count above controls (n=11)
PMN cell count overlapping controls (n=5)Lower SpO2 in group with elevated PMN cell count
(mean±SEM 92.8±0.9 versus 96.6±0.7, p=0.0097) Weight-for-age ns
Respiratory rate ns
Wisconsin chest computed tomography score nsIndependent t-test Sagel [57] P 111 6 months–6 years Neutrophil elastase Lower Shwachman scores in group with detectable free neutrophil elastase activity
(63.5±8.8 versus 69.7±5.5, p<0.0001)Not reported Hull [58] F 32 Presence of inflammation (PMN cell count and IL-8)
Inflammation (n=13)
No inflammation (n=19)Higher lipid hydroperoxide concentration in group with detectable inflammation
(geometric mean 97.7 versus 21.9 μM, p<0.05)
Higher γ-GT in group with detectable inflammation
(geometric mean 104.2 versus 20.2 U·L−1, p<0.05)Independent t-test Data are presented as n, range or mean±sd, unless otherwise stated. Patients grouped by level of inflammation (from BAL biomarkers) differ in clinical presentation (including oxidative stress). P: pooled; F: first; PMN: polymorphonuclear; SpO2: arterial oxygen saturation measured by pulse oximetry; ns: nonsignificant; IL: interleukin; γ-GT: gamma glutamyl transpeptidase.
- Table 5– Responsiveness of bronchoalveolar lavage (BAL) inflammatory markers
First author [ref.] Aliquot fraction analysed Subjects n Age Intervention Results Statistical test Presentation of data PMN cell counts PMN cell % NE IL-6 IL-8 Response to treatment with antibiotics Armstrong [26] F 13 22.1 (14–44) months Eradication of infection
Follow-up BAL at 6–18 monthsDecreased
T1 56.6%
T2 21.2%Decreased
T1 13.6 μg·mL−1
T2 8.5 μg·mL−1Decreased
T1 1782 pg·mL−1
T2 307 pg·mL−1Mean Noah [18] Not specified 15 14–133 months TIS (inhaled 300 mg twice daily, 28 days) (n=6) versus ceftazidime and tobramycin (i.v., 14 days) (n=9)
Follow-up BAL at 4–6 weeksp=0.02
Inhaled +0.9 (0.6–5.4)
Systemic -0.26 (0.03–2.26)ns
Inhaled +5.4 (-11–15.3)
Systemic -7.0 (-34.5–19.1)ns ns Mann–Whitney U-test
Median (range) ratio post versus preGibson [17] Not specified 21 6 months–6 years TIS (inhaled, 28 days) versus placebo
Follow-up BAL at day 28ns ns ns Not reported Gibson [16] Not specified 9 6 months–6 years TIS (28 days)
Follow-up BAL at day 56ns ns ns ns One-sample t-test Gibson [16] Not specified 9 6 months–6 years TIS (28 days)
Follow-up BAL at day 84ns p=0.01
T1 60.5 (29.4)
T2 38.8 (18.2)ns ns One-sample t-test Gibson [16] Not specified 12 6 months–6 years TIS (56 days)
Follow-up BAL at day 112ns p=0.01
T1 53.4 (13.5)
T2 35.4 (20.3)ns ns One-sample t-test Gibson [16] Not specified 8 6 months–6 years TIS (28 days)
Follow-up BAL at day 112ns ns ns ns One-sample t-test Meyer [61] P 11 17–40 years i.v. antibiotics for respiratory exacerbation
BAL start and end of 2 weeks admissionns p=0.017 Not reported Acquisition of infection Armstrong [26] F 8 20 (13–50) months No infection in first BAL (T1) but present in second
Follow-up BAL at 6–18 months (T2)Increased
T1 9.7%
T2 55%Increased
T1 3.3 μg·mL−1
T2 10.3 μg·mL−1Increased
T1 88 pg·mL−1
T2 1206 pg·mL−1Mean Armstrong [13] F 9 27±12 months No infection in first BAL, but present in second
Follow-up BAL at 6–18 monthsns p<0.05
Fold change 2.0 (95% CI 1.2–3.2)p<0.05
Fold change 2.3 (95% CI -1.06–5.2)ANCOVA DNase Ratjen [21] F, P (2 and 3) 29
Treated group5–25 years Treated group (DNase inhaled 2.5 mg once daily, 18 months) versus untreated group (no DNase)
Follow-up BAL at 18 monthsns Unpaired t-test 19
Untreated group5–25 years Paul [19] F, P (2 and 3) 46
Treated group11.3±5.1 years Treated group (DNase inhaled, 36 months) versus untreated group (no DNase)
Follow-up BAL at 18 and 36 monthsIncrease more pronounced in untreated group (p<0.005) than the treated group (p<0.01) Increased in untreated group (p<0.007) but remained stable in treated group Increased in untreated group (p<0.02) but remained stable in treated group Nonparametric repeated measures analyses of variance 39
Untreated group12.2±4.4 years Ratjen [20] F, P (2 and 3) 13
Treated group6–18 years Treated group (DNase inhaled 2.5 mg once daily, 18 months) versus untreated group (no DNase)
Follow-up BAL at 18 monthsDecreased in treated group
-15 (-40–58)
Increased in untreated group
20 (-49–48)
ns between groupsMedian (range)
Mann–Whitney U-test10
Untreated group6–18 years rSLPI McGarvey [6] P 16 27±2 years rSLPI (nebuliser 100 mg, twice daily, 1 week)
Follow-up BAL 12 after the last aerosolp<0.03
Average decrease 68%Not reported All samples are BAL unless stated. PMN: polymorphonuclear; NE: neutrophil elastase; IL: interleukin; F: first; NS: not specified; P: pooled; T1: first paired (range: 6–18 months) BAL sample; T2: second paired (range: 6–18 months) BAL sample; TIS: tobramycin inhalation solution; rSLPI: recombinant secretory leukocyte protease inhibitor.
Additional Files
Supplementary material
Please note: supplementary material is not edited by the Editorial Office, and is uploaded as it has been supplied by the author.
Files in this Data Supplement:
- Supplementary tables - Tables E1-E6
Disclosures
Files in this Data Supplement: