Telomere Length Analysis in Hematologic Disorders

Telomere length of granulocytes and lymphocytes was analyzed in 105 samples from healthy donors as described before [17 (link)], 70 independent patients with AA, and 18 independent patients with DKC. In 5 AA patients and 1 DKC patient, telomere length measurement of the granulocytes was not possible due to insufficient cell number. Flow-FISH for telomere length was performed as described in detail before [12 (link), 59 (link)]. In brief, samples were mixed with a FITC-labeled or Alexa488-labled telomere-specific (CCCTAA)3-peptide nucleic acid FISH probe (Panagene, Daejeon, South Korea) for DNA hybridization followed by DNA counterstaining with LDS 751 (Sigma Aldrich, St. Louis, MO, USA). An FC 500 flow cytometer (Becton Dickinson, Franklin Lakes, NJ, USA) was used for data acquisition. Bovine thymocytes were used as internal controls to calculate telomere length in kilobases and samples were measured in triplicates. The cow thymocytes as well as granulocytes and lymphocytes from human samples were identified based on forward scatter and LDS 751 binding to double-stranded DNA. Telomere age was estimated by linear regression on the age-adjusted samples from healthy donors. Mean average error [MAE] was calculated as follows:
$$\mathrm{MAE}=\frac{1}{n}{\sum }_{i=1}^n\left|x_i\right|$$
with x = delta age. Mean delta age ( $\overline{x}$ ) was calculated as follows:
$$\overline{x}=\frac{1}{n}\sum _{i=1}^n{x}_i$$