Holly D Cox 1 , Geoffrey D Miller 1 , Abhilasha Manandhar 1 , Jacob D Husk 1 , Xuan Jia 2 , James Marvin 3 , Diane M Ward 2 , John Phillips 4 , Daniel Eichner 1 Show Affiliations »
Abstract
BACKGROUND: Immature reticulocytes (IRC) are the first cells to respond to changes in erythropoiesis. For antidoping applications, measurement of IRC may improve detection of blood doping practices. Unfortunately, this small cell population has limited stability in liquid blood samples and is difficult to measure with optimal precision. We developed a method to measure 3 IRC membrane proteins in dried blood spots (DBS) to monitor changes in erythropoiesis. METHODS: DBS spots were washed with buffers to remove soluble proteins, membrane proteins remaining in the spot were digested with trypsin, and one peptide for each protein was measured by LC-MS/MS. IRC protein concentration was determined using a DBS single point calibrator. RESULTS: Intraassay precision for IRC proteins was between 5%-15%. IRC proteins were stable in DBS for 29 days at room temperature. In a longitudinal study of 25 volunteers, the mean intraindividual variation for 3 IRC proteins was 17%, 20%, and 24% from capillary blood DBS. In comparison, the mean longitudinal variation for IRC counts measured on an automated hematology analyzer was 38%. IRC protein concentration from capillary blood DBS correlated well with venous blood DBS protein concentrations. CONCLUSIONS: Measurement of IRC proteins in DBS samples provides a method to measure changes in erythropoiesis with improved analytical sensitivity, stability, and precision. When combined with the inherent advantages of capillary blood collection in the field, this method may substantially improve the detection of blood doping practices. © American Association for Clinical Chemistry 2021. All rights reserved. For permissions, please email: journals.permissions@oup.com.
BACKGROUND: Immature reticulocytes (IRC) are the first cells to respond to changes in erythropoiesis. For antidoping applications, measurement of IRC may improve detection of blood doping practices. Unfortunately, this small cell population has limited stability in liquid blood samples and is difficult to measure with optimal precision. We developed a method to measure 3 IRC membrane proteins in dried blood spots (DBS) to monitor changes in erythropoiesis. METHODS: DBS spots were washed with buffers to remove soluble proteins, membrane proteins remaining in the spot were digested with trypsin, and one peptide for each protein was measured by LC-MS/MS. IRC protein concentration was determined using a DBS single point calibrator. RESULTS: Intraassay precision for IRC proteins was between 5%-15%. IRC proteins were stable in DBS for 29 days at room temperature. In a longitudinal study of 25 volunteers, the mean intraindividual variation for 3 IRC proteins was 17%, 20%, and 24% from capillary blood DBS. In comparison, the mean longitudinal variation for IRC counts measured on an automated hematology analyzer was 38%. IRC protein concentration from capillary blood DBS correlated well with venous blood DBS protein concentrations. CONCLUSIONS: Measurement of IRC proteins in DBS samples provides a method to measure changes in erythropoiesis with improved analytical sensitivity, stability, and precision. When combined with the inherent advantages of capillary blood collection in the field, this method may substantially improve the detection of blood doping practices. © American Association for Clinical Chemistry 2021. All rights reserved. For permissions, please email: journals.permissions@oup.com.
Entities: Chemical
Keywords:
blood doping; dried blood spots; erythropoiesis; protein mass spectrometry; reticulocytes
Mesh： See more »
Year: 2021
PMID： 33993255 DOI： 10.1093/clinchem/hvab058
Source DB: PubMed Journal: Clin Chem ISSN： 0009-9147 Impact factor: 8.327