Measurement of Immature Reticulocytes in Dried Blood Spots by Mass Spectrometry

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.

Tracking immature reticulocyte proteins for improved detection of recombinant human erythropoietin (rhEPO) abuse

“…The average longitudinal precision of CD71/B3, FECH/B3, RET% and IRF%, across 9 subjects was 15 ± 3%, 24 ± 6%, 10 ± 2%, and 19 ± 4%, respectively. The longitudinal precision for RET% and IRF% observed in this study are lower than previously reported 15,27 …”

“…Twenty microliters of K2EDTA venous blood was spotted onto DBS cards and stored at room temperature in a desiccator cabinet. DBS spots were excised from the card and extracted in a 96‐well plate as previously described 15 . Briefly, DBS spots were washed in a series of buffers for 1.5 h to remove the soluble proteins.…”

“…Normalization of immature reticulocyte protein concentration to B3 allows correction for changes in plasma volume, which is a common confounding factor for hematological measurements in athletes 22,23 . In a previous study, CD71/B3 and FECH/B3 in DBS demonstrated good longitudinal precision over 12 weeks 15 . In patients with grade 1 and grade 2 anemia, which often have stimulated erythropoiesis, the concentration of CD71 and FECH was increased 7‐fold and 4‐fold, respectively, when compared to healthy controls 15 .…”

Qualitative and quantitative detection of SARS-CoV-2 antibodies from dried blood spots

“…This is due, at least in part, to ease of collection in pediatric populations, simplicity of transport, and robust testing methods for multiple analytes. While other studies have used DBS cards for research, few have transitioned to translational or clinical applications [13] , [14] , [15] , [16] , [17] , [18] , [19] , [20] . To this end, serological testing from DBS cards has the potential to be useful early in an epidemic or pandemic to better understand both symptomatic and asymptomatic spread of disease in difficult to reach populations particularly those far from testing centers, or in undeveloped countries.…”

“…DBS sampling is currently performed clinically for newborn screening [11] , [12] . DBS sampling has also been proposed for monitoring changes in erythropoiesis and other biologically relevant molecules in the fight against doping in professional sports [13] , [14] , [15] , drug screening [16] , and for the diagnoses of infectious diseases including hepatitis C virus [17] , [18] , cytomegalovirus [19] and HIV [20] . The varied utility of DBS sampling is further reflected by the stability and retrieval of analytes like microRNA [21] , protein [22] and sphingolipids [23] for sensitive molecular assays and mass spectrometry.…”