madman
Super Moderator
Abstract
Background: FDA guidelines limit the use of blood from donors taking testosterone replacement therapy (TRT) to red blood cell (RBC) concentrates, whereas plasma and platelets are discarded. The purpose of this study is to bring awareness to above-average free testosterone concentrations in RBC units from TRT donors.
Study design: We quantified the concentrations of free (bioavailable; pg/ml) and total (protein-bound and free; ng/dl) testosterone in plasma (frozen within 24 h) and supernatants from 42-day stored leukocyte-reduced RBC units from 17 TRT male donors and 17 matched controls (no TRT). Total testosterone concentrations were determined by liquid chromatography with tandem mass spectrometry (LC-MS/MS). Free testosterone concentrations were quantified in the same samples using equilibrium dialysis/LC-MS/MS.
Results: Plasmafree and total testosterone concentrations in TRT donors were 2.9 and 1.8 times higher than that of controls. Total testosterone concentrations in RBC supernatants were about 30% of that of plasma. In contrast, free testosterone concentrations in RBC supernatants were 80%–100% of that of plasma and were significantly (p = .005) higher in TRT compared with controls (252.3 ± 245.3 vs. 103.4 ± 88.2 pg/ml). Supraphysiological free testosterone concentrations (>244 pg/ml) in RBC supernatants were observed in five TRT donors and two control donors.
Conclusions: RBC units from TRT donors may contain supraphysiological concentrations of free testosterone. This may be resolved by avoiding blood collections soon after testosterone dosing and by an enhanced screening of TRT donors. These data establish a rationale for new studies and reexamination of the current guidelines concerning the utilization of blood components from TRT donors.
1 | INTRODUCTION
The clinical use of sex hormones has been historically associated with younger women who may use contraceptive drugs for birth control. In recent years, a different type of sex hormone therapy has gained significant popularity among middle-aged men in the form of testosterone replacement therapy (TRT).1 Soon after, concern has been raised regarding the abuse and health risks associated with exposure to exogenous testosterone.2,3 The rise in TRT popularity has resulted in the appearance of two new populations of prospective blood donors: individuals who presented at blood centers for therapeutic phlebotomy due to testosterone-induced erythrocytosis, and new or returning allogeneic blood donors on testosterone therapy. In response to a growing number of prospective blood donors on prescription testosterone, the U.S Food and Drug Administration (FDA) and the American Association of Blood Banks (AABB) developed policies and procedures to address the safety of blood components collected through therapeutic phlebotomy of individuals with testosterone-induced erythrocytosis. Minutes from a 2014 meeting recommended that only the red blood cells (RBCs) may be distributed for transfusion, whereas the plasma and platelets should be discarded. In addition, donation intervals for such individuals may be shorter than 8 weeks.4 This meeting also addressed the need for studies that will define the concentrations of plasma testosterone that would be acceptable for distribution.
We recently reported that 2.2% of leukocyte-reduced (LR)-RBC units in a regional division of a large blood service organization (Vitalant) were donated by individuals taking testosterone prescription, who was characterized by the high prevalence of obesity, high-frequency blood donations (up to 29 in 24 months compared with 12 in non-TRT control donors), higher blood pressure, and increased hemoglobin concentrations as compared with matched control donors.5 Our evaluations of the quality of stored RBCs from TRT donors (therapeutic and allogeneic blood donations) and matched controls suggested that TRT was associated with changes in RBC metabolism and predisposition to osmotic hemolysis.6 Specifically, we observed significant changes in metabolic pathways related to oxidative stress, free fatty acids, and acyl-carnitines that could partially explain the mechanisms, by which testosterone modulates RBC susceptibility to hemolysis.7 These observations have prompted us to determine the concentrations of testosterone in plasma and LR-RBC units from the same samples characterized in our aforementioned studies. We hypothesized that testosterone concentration in plasma from TRT donors would be higher than that of controls and that most donor testosterone is removed during the production of LR-RBC units. To our surprise, we found that free testosterone (bioavailable form) concentrations in supernatants from 42-day stored RBCs were nearly the same as measured in plasma. Thus, the aim of this study is to bring awareness to this phenomenon that may prompt a reevaluation of current TRT donors' selection criteria and blood utilization and to highlight the need for new studies that will determine the clinical implications of testosterone administered in blood components.
2.2 | Quantification of free and total testosterone
The majority (about 98%) of testosterone in the circulation is in its inactive form and is bound to androgen binding proteins, such as sex hormone-binding globulin (SHBG), whereas free testosterone (about 2%) is the bioavailable form.8 The concentrations of total (protein-bound) testosterone in plasma (frozen within 24 h after blood collection) and supernatants from 7 and 42 days old RBC units (collected after centrifugation of RBC concentrates at 1500x g, 10 min, 18°C) were determined by liquid chromatography with tandem mass spectrometry (LC-MS/MS). Free testosterone concentrations were quantified in the same samples using equilibrium dialysis/LC-MS/MS. All tests were performed in a clinical lab (University of Colorado Hospital). The reference ranges are 47–244 pg/ml for free testosterone and 300–890 ng/dl for total testosterone.
| RESULTS
3.1 | RBC units from TRT donors may contain supraphysiological concentrations of free testosterone
To support the study's hypothesis, we found higher concentrations of free and total testosterone in plasma collected from TRT individuals compared with matched controls. As summarized in Table 1, average plasma-free and total testosterone concentrations were about 2.9 and 1.8 times higher in TRT than in controls, respectively. Average plasma-free testosterone in TRT individuals (305.0 ± 320.7 pg/ml) was above the upper limit of the reference range (244 pg/ml). The large standard deviations observed in plasma-free and total testosterone concentrations reflected the variation in testosterone levels among all individuals and in TRT in particular. In fact, 5 of the 17 tested TRT individuals (about 30%) had supraphysiological concentrations of free and total testosterone levels. In contrast, two individuals (about 12%) from the control group had above-average concentrations of plasma-free and total testosterone.
Evaluation of free testosterone concentrations in supernatants from stored RBCs produced from the same whole blood bags suggested that average free testosterone in the TRT group was about 83% of that of plasma, whereas no change was observed in the control group. In addition, the average free testosterone concentration in RBC supernatants from TRT donors was about 2.5 times higher (p = .005) than that of controls (252.3 ± 245.3 pg/ml vs. 103.4 ± 88.2 pg/ml; Table 1). In comparison, lower free testosterone concentrations were measured in RBC supernatants from two female donors (5.4 and 8.1 pg/ml), suggesting that the concentrations observed in RBCs from male donors were not due to an assay artifact. Average total testosterone concentrations in RBC supernatants from all donors were about 30% of that of plasma, suggesting that the majority (about 70%) of total testosterone was removed during the manufacturing of RBC concentrates.
As shown in Figure 1A, the difference between plasma and RBC supernatant concentrations of free testosterone was larger in individuals with supraphysiological testosterone than those with reference range testosterone levels. Correlation analysis (Spearman's r) revealed significant (p < .0001) and strong (r = .9832) association between plasma and RBC supernatant free testosterone concentrations (Figure 1B). A decrease (51%–83%) in total testosterone concentrations between plasma and RBC supernatant was observed in all samples (Figure 1C). Similar to free testosterone, we observed a good correlation (Spearman's r = .8655, p < .0001) in total testosterone concentrations between plasma and RBC supernatant (Figure 1D). However, none of the total testosterone concentrations found in the RBC supernatants were above the reference range.
Considering these observations, we asked whether free testosterone concentrations in RBC supernatants vary throughout cold storage. To answer this question, we quantified the concentrations of free testosterone in 7-day-old stored RBCs in a subset of the donors (two TRT and two matched controls) and found that these concentrations (61.3 ± 23.1 pg/ml) were comparable, although somewhat lower (p = .04), to those measured at day 42 of storage (69.3 ± 25.7 pg/ml) or in plasma (66.9 ± 29.8 pg/ml) (Figure 2)
4 | DISCUSSION
4.1 | RBCs are possible carriers of free testosterone
The unanticipated presence of free testosterone in RBC unit supernatants is puzzling and raises questions about its distribution in whole blood. If the majority of free testosterone is localized in plasma (as opposed to blood cells), then the removal of plasma during the manufacturing of LR-RBC units would significantly reduce its concentration in RBC units as in the case of total testosterone. This assumption may have driven current blood banking policies to restrict blood utilization from TRT donors to LR-RBCs and to discard plasma and platelets. Furthermore, the low concentration of residual donor plasma (about 5%)9 found in LRRBC units cannot explain the high concentrations of free testosterone, which are similar to what we observed in the plasma bags. One explanation relies on the unique characteristic of RBCs to transport and deliver a variety of drug formulations including nanoparticles.10 This phenomenon was given the term “red blood cell hitchhiking.” 11 A study that evaluated the permeability of RBCs to radio-labeled sex hormones including testosterone suggested that RBCs may contribute to 5%–15% of sex hormone transport in the circulation.12 Similar to other sex steroids, (free) testosterone is lipophilic in nature and capable of diffusing into target cells.13 Thus, it is possible that the free testosterone concentrations found in our tested RBC units reflect a mechanism by which testosterone is transiently “hosted” in RBCs that may contribute to its transport to targeted tissues. In contrast, protein-bound testosterone is incapable of freely diffusing across cell membranes, which may explain why 70% of it was removed during the production of LRRBC units. The remaining protein-bound testosterone may be present in the residual plasma or enclosed by RBCs. These, of course, are only assumptions that require further validation.
4.2 | Do supraphysiological concentrations of free testosterone in RBC units pose a risk to transfusion recipients?
4.3 | Recommended actions for reevaluation of policies concerning blood donors with prescription testosterone
In conclusion, our findings established a rationale for reexamination of the current FDA and AABB guidelines concerning the safety and utilization of blood components from TRT donors. If concerns about supraphysiological testosterone have driven the decision to reject platelets and plasma-derived from TRT blood donations, then LR-RBC units are no exception with regard to free testosterone. If deemed safe, blood utilization from such individuals can be expanded to include platelets and plasma products. This is important in times of blood shortage as recently experienced during the outbreak of COVID19 and in light of the rise in TRT popularity among men, which is expected to further increase the number of blood donors on this therapy.
Background: FDA guidelines limit the use of blood from donors taking testosterone replacement therapy (TRT) to red blood cell (RBC) concentrates, whereas plasma and platelets are discarded. The purpose of this study is to bring awareness to above-average free testosterone concentrations in RBC units from TRT donors.
Study design: We quantified the concentrations of free (bioavailable; pg/ml) and total (protein-bound and free; ng/dl) testosterone in plasma (frozen within 24 h) and supernatants from 42-day stored leukocyte-reduced RBC units from 17 TRT male donors and 17 matched controls (no TRT). Total testosterone concentrations were determined by liquid chromatography with tandem mass spectrometry (LC-MS/MS). Free testosterone concentrations were quantified in the same samples using equilibrium dialysis/LC-MS/MS.
Results: Plasmafree and total testosterone concentrations in TRT donors were 2.9 and 1.8 times higher than that of controls. Total testosterone concentrations in RBC supernatants were about 30% of that of plasma. In contrast, free testosterone concentrations in RBC supernatants were 80%–100% of that of plasma and were significantly (p = .005) higher in TRT compared with controls (252.3 ± 245.3 vs. 103.4 ± 88.2 pg/ml). Supraphysiological free testosterone concentrations (>244 pg/ml) in RBC supernatants were observed in five TRT donors and two control donors.
Conclusions: RBC units from TRT donors may contain supraphysiological concentrations of free testosterone. This may be resolved by avoiding blood collections soon after testosterone dosing and by an enhanced screening of TRT donors. These data establish a rationale for new studies and reexamination of the current guidelines concerning the utilization of blood components from TRT donors.
1 | INTRODUCTION
The clinical use of sex hormones has been historically associated with younger women who may use contraceptive drugs for birth control. In recent years, a different type of sex hormone therapy has gained significant popularity among middle-aged men in the form of testosterone replacement therapy (TRT).1 Soon after, concern has been raised regarding the abuse and health risks associated with exposure to exogenous testosterone.2,3 The rise in TRT popularity has resulted in the appearance of two new populations of prospective blood donors: individuals who presented at blood centers for therapeutic phlebotomy due to testosterone-induced erythrocytosis, and new or returning allogeneic blood donors on testosterone therapy. In response to a growing number of prospective blood donors on prescription testosterone, the U.S Food and Drug Administration (FDA) and the American Association of Blood Banks (AABB) developed policies and procedures to address the safety of blood components collected through therapeutic phlebotomy of individuals with testosterone-induced erythrocytosis. Minutes from a 2014 meeting recommended that only the red blood cells (RBCs) may be distributed for transfusion, whereas the plasma and platelets should be discarded. In addition, donation intervals for such individuals may be shorter than 8 weeks.4 This meeting also addressed the need for studies that will define the concentrations of plasma testosterone that would be acceptable for distribution.
We recently reported that 2.2% of leukocyte-reduced (LR)-RBC units in a regional division of a large blood service organization (Vitalant) were donated by individuals taking testosterone prescription, who was characterized by the high prevalence of obesity, high-frequency blood donations (up to 29 in 24 months compared with 12 in non-TRT control donors), higher blood pressure, and increased hemoglobin concentrations as compared with matched control donors.5 Our evaluations of the quality of stored RBCs from TRT donors (therapeutic and allogeneic blood donations) and matched controls suggested that TRT was associated with changes in RBC metabolism and predisposition to osmotic hemolysis.6 Specifically, we observed significant changes in metabolic pathways related to oxidative stress, free fatty acids, and acyl-carnitines that could partially explain the mechanisms, by which testosterone modulates RBC susceptibility to hemolysis.7 These observations have prompted us to determine the concentrations of testosterone in plasma and LR-RBC units from the same samples characterized in our aforementioned studies. We hypothesized that testosterone concentration in plasma from TRT donors would be higher than that of controls and that most donor testosterone is removed during the production of LR-RBC units. To our surprise, we found that free testosterone (bioavailable form) concentrations in supernatants from 42-day stored RBCs were nearly the same as measured in plasma. Thus, the aim of this study is to bring awareness to this phenomenon that may prompt a reevaluation of current TRT donors' selection criteria and blood utilization and to highlight the need for new studies that will determine the clinical implications of testosterone administered in blood components.
2.2 | Quantification of free and total testosterone
The majority (about 98%) of testosterone in the circulation is in its inactive form and is bound to androgen binding proteins, such as sex hormone-binding globulin (SHBG), whereas free testosterone (about 2%) is the bioavailable form.8 The concentrations of total (protein-bound) testosterone in plasma (frozen within 24 h after blood collection) and supernatants from 7 and 42 days old RBC units (collected after centrifugation of RBC concentrates at 1500x g, 10 min, 18°C) were determined by liquid chromatography with tandem mass spectrometry (LC-MS/MS). Free testosterone concentrations were quantified in the same samples using equilibrium dialysis/LC-MS/MS. All tests were performed in a clinical lab (University of Colorado Hospital). The reference ranges are 47–244 pg/ml for free testosterone and 300–890 ng/dl for total testosterone.
| RESULTS
3.1 | RBC units from TRT donors may contain supraphysiological concentrations of free testosterone
To support the study's hypothesis, we found higher concentrations of free and total testosterone in plasma collected from TRT individuals compared with matched controls. As summarized in Table 1, average plasma-free and total testosterone concentrations were about 2.9 and 1.8 times higher in TRT than in controls, respectively. Average plasma-free testosterone in TRT individuals (305.0 ± 320.7 pg/ml) was above the upper limit of the reference range (244 pg/ml). The large standard deviations observed in plasma-free and total testosterone concentrations reflected the variation in testosterone levels among all individuals and in TRT in particular. In fact, 5 of the 17 tested TRT individuals (about 30%) had supraphysiological concentrations of free and total testosterone levels. In contrast, two individuals (about 12%) from the control group had above-average concentrations of plasma-free and total testosterone.
Evaluation of free testosterone concentrations in supernatants from stored RBCs produced from the same whole blood bags suggested that average free testosterone in the TRT group was about 83% of that of plasma, whereas no change was observed in the control group. In addition, the average free testosterone concentration in RBC supernatants from TRT donors was about 2.5 times higher (p = .005) than that of controls (252.3 ± 245.3 pg/ml vs. 103.4 ± 88.2 pg/ml; Table 1). In comparison, lower free testosterone concentrations were measured in RBC supernatants from two female donors (5.4 and 8.1 pg/ml), suggesting that the concentrations observed in RBCs from male donors were not due to an assay artifact. Average total testosterone concentrations in RBC supernatants from all donors were about 30% of that of plasma, suggesting that the majority (about 70%) of total testosterone was removed during the manufacturing of RBC concentrates.
As shown in Figure 1A, the difference between plasma and RBC supernatant concentrations of free testosterone was larger in individuals with supraphysiological testosterone than those with reference range testosterone levels. Correlation analysis (Spearman's r) revealed significant (p < .0001) and strong (r = .9832) association between plasma and RBC supernatant free testosterone concentrations (Figure 1B). A decrease (51%–83%) in total testosterone concentrations between plasma and RBC supernatant was observed in all samples (Figure 1C). Similar to free testosterone, we observed a good correlation (Spearman's r = .8655, p < .0001) in total testosterone concentrations between plasma and RBC supernatant (Figure 1D). However, none of the total testosterone concentrations found in the RBC supernatants were above the reference range.
Considering these observations, we asked whether free testosterone concentrations in RBC supernatants vary throughout cold storage. To answer this question, we quantified the concentrations of free testosterone in 7-day-old stored RBCs in a subset of the donors (two TRT and two matched controls) and found that these concentrations (61.3 ± 23.1 pg/ml) were comparable, although somewhat lower (p = .04), to those measured at day 42 of storage (69.3 ± 25.7 pg/ml) or in plasma (66.9 ± 29.8 pg/ml) (Figure 2)
4 | DISCUSSION
4.1 | RBCs are possible carriers of free testosterone
The unanticipated presence of free testosterone in RBC unit supernatants is puzzling and raises questions about its distribution in whole blood. If the majority of free testosterone is localized in plasma (as opposed to blood cells), then the removal of plasma during the manufacturing of LR-RBC units would significantly reduce its concentration in RBC units as in the case of total testosterone. This assumption may have driven current blood banking policies to restrict blood utilization from TRT donors to LR-RBCs and to discard plasma and platelets. Furthermore, the low concentration of residual donor plasma (about 5%)9 found in LRRBC units cannot explain the high concentrations of free testosterone, which are similar to what we observed in the plasma bags. One explanation relies on the unique characteristic of RBCs to transport and deliver a variety of drug formulations including nanoparticles.10 This phenomenon was given the term “red blood cell hitchhiking.” 11 A study that evaluated the permeability of RBCs to radio-labeled sex hormones including testosterone suggested that RBCs may contribute to 5%–15% of sex hormone transport in the circulation.12 Similar to other sex steroids, (free) testosterone is lipophilic in nature and capable of diffusing into target cells.13 Thus, it is possible that the free testosterone concentrations found in our tested RBC units reflect a mechanism by which testosterone is transiently “hosted” in RBCs that may contribute to its transport to targeted tissues. In contrast, protein-bound testosterone is incapable of freely diffusing across cell membranes, which may explain why 70% of it was removed during the production of LRRBC units. The remaining protein-bound testosterone may be present in the residual plasma or enclosed by RBCs. These, of course, are only assumptions that require further validation.
4.2 | Do supraphysiological concentrations of free testosterone in RBC units pose a risk to transfusion recipients?
4.3 | Recommended actions for reevaluation of policies concerning blood donors with prescription testosterone
In conclusion, our findings established a rationale for reexamination of the current FDA and AABB guidelines concerning the safety and utilization of blood components from TRT donors. If concerns about supraphysiological testosterone have driven the decision to reject platelets and plasma-derived from TRT blood donations, then LR-RBC units are no exception with regard to free testosterone. If deemed safe, blood utilization from such individuals can be expanded to include platelets and plasma products. This is important in times of blood shortage as recently experienced during the outbreak of COVID19 and in light of the rise in TRT popularity among men, which is expected to further increase the number of blood donors on this therapy.
