Telomere length measurement

We extracted DNA from patient pre-HCT peripheral blood mononuclear cells or whole blood using the QIAamp Maxi Kit procedure (Qiagen Inc., Valencia, CA). TL was measured by monoplex qPCR at the NCI Cancer Genomics Research laboratory after adapting published methods,(Callicott and Womack 2006, Cawthon 2002) as described previously (Gadalla, et al 2016a). Briefly, relative telomere length (RTL) was calculated as the ratio of telomere (T) signal concentration to that of a single copy gene (RPLP0, also termed 36B4; S); raw measurements were then standardized using internal quality-controlled calibrator samples replicated within each plate. All telomeric and RPLP0/36B4 results were measured in triplicate and their average was used for all calculations. Final RTL values were log transformed to ensure normality. RTL for SAA patients and healthy donors were measured in two sets. The mean coefficient of variations (CV) for telomeric measures (T) was 0.45%, and for single copy gene (RPLP0/36B4) was 0.4% in all samples, and 5.23% for the standardized T/S measurement from replicate samples. Laboratory personnel were blinded to patient characteristics and outcomes.