Podium Presentation in Room 5 on Wednesday at 14:00 (Chair: Andrew Hoofnagle)
Authors: Holly D. Cox, Geoff D. Miller, Abhilasha Manandhar, Jacob Husk, Daniel Eichner
Immature reticulocytes are the first cells to respond to erythropoietic signals. As such, they are good indicators of bone marrow recovery after transplantation, chemotherapy, or treatment with erythropoiesis-stimulating agents. For anti-doping applications, immature reticulocyte counts may be a good biomarker for detection of blood doping practices. Due to the low number of immature reticulocytes in circulation, automated hematology analyzers cannot measure them with good precision. We have developed a method to measure immature reticulocyte membrane proteins in dried blood spots (DBS). Collection of DBS samples will allow more frequent blood collections in the field, in remote locations, and under un-announced out-of-competition testing conditions, which may improve detection of blood doping practices. However, it is unclear if capillary blood collection methods and dried blood spot samples have sufficient precision to provide biologically meaningful data.
We compared the longitudinal precision of immature reticulocyte proteins in DBS from capillary blood and venous blood from 25 individuals for 8 weeks. Two different capillary blood collection devices were compared. Longitudinal precision in DBS was compared to precision of the immature reticulocyte cell counts on an automated hematology analyzer. Finally, the immature reticulocyte proteins in DBS were measured after administration of recombinant human erythropoietin (EPO).
The immature reticulocyte membrane proteins, CD71, ferrochelatase, and band 3 were measured in DBS after washing the spots in 3 buffers to remove soluble proteins. The membrane proteins remaining in the spot were digested with trypsin for 2 hrs after addition of cleavable, stable isotope labeled peptide internal standards. Peptides released from the spot were measured by a parallel reaction monitoring method on a QExactive Plus mass spectrometer. One peptide was measured for each protein. A DBS single point calibrator was used to calculate concentrations.
CD71 concentration in DBS correlates well with immature reticulocyte cell number measured on an automated hematology analyzer, r2 0.8875. The average longitudinal CV% for CD71 and ferrochelatase was 20% and 24%, respectively, from venous blood DBS. This is a significant improvement over the average longitudinal precision observed on the automated hematology analyzer of 41%. In capillary blood DBS, CD71 longitudinal precision was similar to venous DBS when collected by TAP or TASSO devices at 20% and 17%, respectively. After EPO administration, CD71 and ferrochelatase increased 5-fold and 3-fold, respectively, which is a significant improvement over the current method.
Measurement of CD71, ferrochelatase, and band 3 by mass spectrometry provides better longitudinal precision to monitor immature reticulocyte activity. This method may significantly improve the detection of blood doping practices.
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