Table 1.
Demographic and clinical characteristics of participants of this study.
Table 2.
Comparison between telomere length methods: linear regression and Bland-Altman analysis.
Figure 1.
Comparison between flow-FISH and TRF analysis of leukocyte telomere length in healthy individuals and patients with bone marrow failure or idiopathic pulmonary fibrosis: linear correlation and Bland-Altman agreement.
Telomere length of 70 healthy individuals was measured by flow-FISH and Southern blot: (A) Linear regression plots; solid line represents the data best fit (R2 = 0.60); and (B) Bland-Altman plot for agreement analysis of flow-FISH (kb) and TRF analysis (kb). The bias±SD was 0.17±1.03 and limits of agreement (LoA) ranged from −1.88 to 2.24 kb. Telomere length of 51 patients was measured by both methods: (C) Linear regression plots; solid line represents the data best fit (R2 = 0.51); and (D) Bland-Altman plot for agreement analysis of flow-FISH (kb) and TRF analysis (kb). The bias±SD was zero ±1.21 and the LoA ranged from −2.41 to 2.41. Measurements were represented in kilobases.
Figure 2.
Comparison between qPCR and TRF analysis of leukocyte telomere length in the healthy individuals and patients with bone marrow failure or idiopathic pulmonary fibrosis: linear correlation and Bland-Altman agreement.
Telomere length from 70 healthy individuals was measured by qPCR and Southern blot: (A) Linear regression plots of qPCR (T/S ratio) x TRF analysis (kb) measurements; solid line represents the data best fit (R2 = 0.35); and (B) Bland-Altman plot for agreement analysis of qPCR (kb) and TRF analysis (kb). The bias±SD was 0.78±1.34 and limits of agreement (LoA) ranged from −1.90 to 3.47. Telomere length of 51 patients was measured by both methods: (C) Linear regression plots qPCR (T/S ratio) x TRF analysis (kb) measurements; solid line represents the data best fit (R2 = 0.20) and (D) Bland-Altman plot for agreement analysis of qPCR (kb) and TRF analysis (kb). The bias±SD was 1.15±1.49 and the LoA ranged from −1.84 to 4.14.
Figure 3.
Comparison between qPCR and flow-FISH of leukocyte telomere length in the healthy individuals and patients with bone marrow failure or idiopathic pulmonary fibrosis: linear correlation and Bland-Altman agreement.
Telomere length of 70 healthy individuals was measured by qPCR and flow-FISH: (A) Linear regression plots of qPCR (T/S ratio) x flow-FISH (kb) measurements; solid line represents the data best fit (R2 = 0.33); and (B) Bland-Altman plot for agreement analysis of qPCR (kb) and flow-FISH (kb). The bias±SD was -0.6±1.27 and limits of agreement (LoA) ranged from −3.16 to 1.94. Telomere length of 51 patients was measured by both methods: (C) Linear regression plots of qPCR (T/S ratio) x flow-FISH (kb) measurements and solid line represents the data best fit (R2 = 0.10). (D) Bland-Altman plot for agreement analysis of qPCR (kb) and flow-FISH (kb). The bias±SD was −1.15±1.65 and the LoA ranged from −4.45 to 2.15.
Figure 4.
Variability analysis of flow-FISH and qPCR.
(A) Intra-assay variation ±95% confidence interval. Flow-FISH, CV = 10.8%; qPCR, CV = 9.5%; p = 0.35. (B) Inter-assay variation ±95% confidence interval. Flow-FISH, CV = 9.5%; qPCR, CV = 16%; p = 0.02. (C) Bland-Altman plot for flow-FISH: mean difference between two independent measurements of 23 samples by their average; the bias was −0.08±1.07 and limits of agreement ranged from −2.23 to 2.05. (D) Bland-Altman plot for qPCR: mean difference between two independent measurements of 57 samples by their average; the bias was −0.37±1.7 and limits of agreement ranged from −3.02 to 3.78. For Bland-Altman analysis, telomere length measurements were represented in kilobases.
Figure 5.
Telomere length according to age in patients with bone marrow failure or idiopathic pulmonary fibrosis measured by Southern blot, flow-FISH and qPCR.
Lines represent the 1st, 10th, 50th, 90th, 99th percentiles of telomere length of healthy controls (Figure S1). (A) Telomere length measurement by Southern blot was used to define patients with dyskeratosis congenita or BMF/IPF with telomerase mutation who had very short telomeres (<1st percentile; represented by red dots), patients with short telomeres (<10th percentile; represented by black dots) and patients with normal telomeres (above 10th percentile; circles). (B) Telomere length measurement by flow-FISH of patients with very short, short and normal telomeres defined by Southern blot. (C) Telomere length measurement by qPCR of patients with very short, short and normal telomeres defined by Southern blot.