Holly Cox (Presenter)
Sports Medicine Research and Testing Laboratory
Bio: Holly Cox is the Senior Research Scientist at the Sports medicine Research and Testing Laboratory. Her research focuses on the development of mass spectrometry-based protein quantitation methods for antidoping applications. She has developed methods for the detection of growth hormone secretagogues and releasing hormones, insulin analogues, insulin-like growth factor-1, and blood doping practices.
Authorship: Holly D. Cox1; Geoff D. Miller1, Auriella Lai1, Laura Garvican-Lewis2, Daniel Eichner1
1. Sports Medicine Research and Testing Laboratory, Salt Lake City, UT 84108, 2. Australian Institute of Sport, Canberra, Australia
Membrane proteins include several important drug targets, transporters, receptors, and cell differentiation markers. We have developed a method to enrich for membrane proteins and reduce matrix interference in dried blood spot (DBS) samples. The method was applied to the measurement of four cluster of differentiation (CD) proteins, CD71, Band3, CD45, and CD41, which serve as blood cell-specific markers. The DBS method was validated and tested in subjects under several conditions including iron deficient anemia, after blood withdrawal, after autologous blood transfusion, and at high altitude, with and without iron supplementation.
The dried blood spot (DBS) matrix may have utility for applications where samples are collected in the field, when timely shipment of unstable liquid blood samples is not possible, or for collection of longitudinal data. These conditions often apply in an antidoping setting where samples are collected at sporting events and the blood data is longitudinally monitored over time for each athlete. Unfortunately, protein measurement in DBS is hindered by high abundance proteins and matrix interference. To address these issues, we have developed a DBS method to enrich for integral membrane proteins and reduce matrix interference.(1) The method extracts DBS spots in a series of buffers to remove soluble proteins while leaving cell membranes and membrane proteins on the spot. After extraction, the remaining membrane proteins are digested with trypsin and the released peptides are quantified. This method was applied to the measurement of four cluster of differentiation (CD) proteins, CD71, Band3, CD45, and CD41, which serve as cell-specific markers of immature reticulocytes (IRC), red blood cells (RBC), white blood cells (WBC), and platelets (PLT), respectively. The DBS method was validated and compared to blood cell counts obtained with an automated hematology analyzer.
The DBS method was further tested for the detection of blood doping practices in sport. The current method used by antidoping laboratories measures hemoglobin concentration and reticulocyte% (Ret%), which are sensitive to changes in erythropoiesis and able to detect several forms of blood manipulation including blood transfusions and erythropoiesis stimulation agents. The DBS method was compared to the current method and tested in subjects under several conditions relevant to antidoping: normal and anemic subjects, after blood withdrawal, after autologous blood transfusion, and at high altitude ± iron supplementation.
DBS samples were obtained from subjects in the following categories: after blood withdrawal, after blood transfusion, and at high altitude. DBS spots were washed in a series of 3 buffers and the remaining membrane proteins were digested with trypsin for 2 hours at 60 °C. Peptides released from the spot were measured at a resolution of 70,000 FWHM on a QExactive Plus (Thermo-Fisher) in parallel reaction monitoring mode. One peptide was measured for each protein, CD71, Band3, CD45 and CD41. Cleavable, stable isotope labeled peptides were used as internal standards. A hematocrit curve at 35%, 45%, 55% was used as a calibration curve to calculate RBC, WBC and PLT from peptide peak area ratios. Recombinant CD71, soluble domain, spiked into rat DBS extracts at three concentrations was used to calculate CD71 concentration.
The DBS extraction and membrane protein enrichment method allowed the measurement of CD71 on immature reticulocytes present in blood at 10e3 cells per microliter, while in a mixture of RBC, present in blood at 10e6 cells per microliter. The extraction steps successfully removed soluble proteins, including serum transferrin receptor, which could interfere with measurement of CD71. DBS cell counts demonstrated good correlation with flow cytometry and hematology analyzer results.
Using the DBS method, it was possible to estimate the average CD protein concentration per cell and calculate the interindividual variation. Band3 and CD45 displayed very low interindividual variation, thus allowing accurate cell counts using a human hematocrit curve. However, for CD71, the total concentration is dependent not only upon IRC cell number, but also upon the maturation state of the cells. Therefore, it is more accurate to measure CD71 concentration directly rather than calculate IRC number. CD71 protein concentration was measured using a recombinant standard of the soluble domain spiked into rat DBS extracts. Using this calibration curve, it was possible to longitudinally monitor CD71 concentrations over time, which is not possible using standard flow cytometry methods. Longitudinal measurement of CD71 concentration was stable over an 8-week period when tested in six individuals. Additionally, CD71 concentration per cell was significantly higher in subjects with iron deficient anemia than in normal subjects. This is consistent with the regulation of CD71 by intracellular iron, which increases CD71 concentration under low iron conditions.
For detection of blood doping practices in sport, the current method was compared to the DBS method in an autologous transfusion study. In this study, 26 subjects had 1 unit of blood removed and stored for 21 days. After storage, 15 subjects received blood transfusion and 11 subjects received saline transfusion. The response of selected blood parameters was monitored for 5 weeks post-transfusion. Reticulocyte% was measured using a hematology analyzer and compared to the CD71/Band3 ratio measured in DBS. After blood transfusion, negative feedback inhibition causes a decrease in erythropoiesis which was observed as a decrease in Ret% at days 4-20 post-transfusion. The CD71/Band3 ratio decreased in a similar manner at days 5-20 post-transfusion. The magnitude of the response was larger in the CD71/Band3 ratio than for Ret%, indicating that the DBS method may improve the sensitivity of the test. After blood withdrawal, there was a large increase in the CD71/Band3 ratio in iron deficient/anemic subjects that was not observed in normal subjects.
Finally, Ret% and the CD71/Band3 ratio response was compared under conditions of high altitude ± iron supplementation, designed to simulate the live high train low strategy adopted by many athletes. After 2 weeks of high altitude, Ret% was increased to 112%, 136% and 152% of baseline after supplementation with placebo, oral iron, and iv iron, respectively. Thus, the Ret% response was increased by iron supplementation. In contrast, the CD71/Band3 ratio increased to 123%, 126%, and 119% after supplementation with placebo, oral iron, and iv iron, respectively. Thus, the CD71/Band3 ratio was not increased by iron supplementation. The response may be consistent with an intracellular iron-regulated decrease in CD71 protein.
Conclusions & Discussion
CD proteins are a class of protein markers used to characterize cell types and disease states in blood. The described method provides a way to measure the concentration of CD proteins and count cell types in DBS, which may have several applications. The method allows longitudinal measurement of CD proteins, which is not possible by flow cytometry methods. Use of the DBS matrix allows application of this method to samples collected outside of the clinic and in remote locations.
For antidoping applications, the CD71/Band3 ratio displayed good agreement with Ret%. Differences may be observed between Ret% and CD71/Band3 ratio in iron deficient anemia and with iron supplementation. Use of the DBS method for detection of blood doping practices will increase the testing frequency and may improve the sensitivity of the current method.
References & Acknowledgements:
1. Cox HD, Eichner D. A mass spectrometry method to measure membrane proteins in dried blood spots for the detection of blood doping practices in sport. Anal.Chem (2017) doi: 10.1021/acs.analchem.7b02492. [Epub ahead of print]
This work was supported by a grant from the Partnership for Clean Competition.
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