While spaceflight anemia has been consistently reported post-flight and during short-duration flight [3, 5], little is known about the in-flight condition during long-duration missions. In this study, we observed statistically significant elevations in the concentrations of RBCs, platelets, and hemoglobin, and we interpret an apparent increase in hematocrit at multiple time points during long-duration spaceflight.
The alterations associated with spaceflight observed in this study are in accordance with previous findings of elevated RBC indices in-flight. RBC concentration, hemoglobin, and hematocrit have been shown to be elevated during the first few days of flight [2, 4, 11]; however, here we show that RBC concentration remains elevated even after the initial period of adaptation to microgravity. Although previous findings suggest that RBC mass is decreased in association with spaceflight [2,3,4, 7], alterations in cell mass and concentration need not track together. While the observed elevations in RBC concentration and hematocrit may simply be due to greater losses in plasma volume than in RBC mass, it is possible that RBC mass is partially restored as the body adjusts to the absence of gravity as flight duration extends, and the losses in RBC mass are less severe during long-duration spaceflight. In a review of literature on RBC mass and spaceflight, Tavassoli et al.  noted that in the first 3 weeks of flight, length of flight and losses in RBC mass were positively correlated, with greater losses in RBC mass occurring in longer flights; however, in the studies performed on the longer-duration Skylab 2, 3 and 4 missions (28, 59, and 84 days, respectively), the longer missions were actually associated with smaller decreases in RBC mass [3, 7]. Therefore it has been previously postulated that during prolonged exposure to microgravity a new RBC mass homeostasis is reached, and the early reductions in RBC mass are abrogated [5, 12].
The observed reduction in MCH late in-flight may be reflected in the relationship between RBC concentration and hemoglobin, as RBC concentration remained elevated throughout the flight while hemoglobin was significantly elevated only early in the flight. A reduced requirement for oxygen-carrying capabilities and easier delivery of oxygen to tissues while in microgravity may drive some of these changes [5, 6].
Previous post-flight findings are varied, as both elevations [7, 13] and depressions [7, 11] in RBC count, hemoglobin, and hematocrit have been reported. Here we found significant post-flight decreases in hematocrit and MCV, while all other parameters rapidly returned to baseline upon re-entry. Interestingly, immediately after the 28-day Skylab 2 mission RBC count, hemoglobin concentration and hematocrit fell below pre-flight values, and while RBC count had recovered by day 7 post-flight, hematocrit and hemoglobin concentration were still below pre-flight levels at 18 days post-flight . In contrast, on the Skylab 3 and 4 missions (59 and 84 days, respectively) RBC count, hemoglobin concentration, and hematocrit were elevated immediately upon landing, but subsequently began to decline and were significantly lower than pre-flight values 3 days after landing, returning to normal in the 3 week testing period following the flights . With the dependence of these indices on plasma volume, the timing of the sample and the conditions of the return may have a large impact. Both dehydration and plasma volume shifts upon re-entry into gravity can significantly affect these parameters. Plasma volume has been shown to be rapidly restored upon re-entry [14, 15], which may account for the rapid return to baseline values of RBC count observed in this study, given the in-flight elevations in these parameters; however, without an accurate measure of plasma volume, it is difficult to make any conclusive statements. Additional sampling between the R + 0 and the R + 30 samples may be beneficial in determining the erythrokinetics post-flight. Depressions in RBC count, hemoglobin concentration, and hematocrit in the weeks after spaceflight were reported after the Skylab missions and by others [2, 7, 11, 14] and were interpreted as potential depressions in red blood cell mass during spaceflight that were slower to recover upon return to Earth than the depressions in plasma volume. Monitoring the RBC indices in the days following flight in the current study would have provided interesting information, given the observed in-flight elevations, and not depressions, in various hematologic indices.
Little data exist regarding in-flight platelet concentrations ; however, the reports that do exist suggest that microgravity and simulated microgravity actually induce a state of thrombocytopenia [16, 17]. In contrast, the elevations in platelet concentration observed in this investigation at the early and mid-flight time points may be due to reductions in plasma volume without any true increase in platelet numbers. The gradual return toward baseline of platelet concentration over the course of the 6-month mission may be indicative of a homeostatic mechanism that serves to counteract elevations in platelet concentration resulting from reduced plasma volume. Interestingly, BE Crucian, SR Zwart, S Mehta, P Uchakin, HD Quiriarte, D Pierson, CF Sams and SM Smith  recently reported that plasma thrombopoietin, which stimulates platelet production and is generally elevated when platelet levels are low, was elevated throughout 6-months of orbital spaceflight; however, vascular endothelial growth factor (VEGF) and C-X-C motif chemokine 5 (CXCL5), both of which are platelet-derived and positively correlated with platelet concentration [19, 20], were also elevated throughout the 6-month missions . The elevations in plasma VEGF and CXCL5 , in conjunction with the finding that platelet concentration was also elevated, appears to indicate that long-duration spaceflight does not induce thrombocytopenia; however, the discrepant finding that thrombopoietin was also elevated  warrants further investigation.
Although the performance of a CBC on samples collected during spaceflight generated novel information, these findings must be interpreted with caution. The cellular concentrations are dependent on plasma volume, and therefore the observed elevations may be influenced by reductions in plasma volume without any real increase in cellular mass. Indeed, plasma volume has been shown to decrease by approximately 17% within the first 24 h of spaceflight ; however, like changes in RBC mass, the alterations in plasma volume have been primarily observed during short-duration flight or post-flight, and little evidence exists describing changes in plasma volume during long-duration spaceflight. The reductions in plasma volume observed between flight days 8 and 12 by Alfrey et al. , while still significant, were smaller than the reductions observed on the first flight day, indicating there may be a continued trend toward plasma volume recovery as time on board the ISS progresses. In a comparison of short and long-duration flights, the average loss in plasma volume for 5 long-duration astronauts was marginally lower than the average loss in 29 short-duration astronauts, though this was not statistically significant . To fully interpret the alterations presented in the current study, plasma volume must also be assessed during long-duration space flight.
The measurement of erythropoietin (EPO) in-flight would also aid in the interpretation of the reported findings; unfortunately EPO was not determined as part of the parent immune investigations. EPO controls RBC mass by regulating the rate of division of RBC progenitors in the bone marrow, and it has also been postulated to play a role in the neocytolysis process by which newly released RBCs are selectively destroyed upon entering into microgravity [12, 15, 22]. EPO has been shown to be reduced early in-flight but elevated following short-duration flight , indicating that homeostatic mechanisms attempt to reduce RBC mass upon entering microgravity and restore it upon landing. However, to our knowledge, EPO has not been measured during long-duration flight. The measurement of EPO in future studies of prolonged spaceflight may help to explain the present findings of elevated RBC count throughout long-duration flight.
The delay in processing for the in-flight blood samples is also a limitation of the study. RBC, hemoglobin and platelet concentration have all been shown to be stable for up to 72 h when blood samples collected with EDTA are stored at 4 °C ; however, blood samples for our investigations were returned at ambient temperature. Despite the recommendations that samples be refrigerated, results of the stability tests indicate that RBC count, hemoglobin concentration, MCH values, and platelets remain stable for at least 48 h, even at room temperature. The elevations in hematocrit and MCV reported here are in accordance with other study findings. MCV begins to increase within 6–12 h of blood collection, which, in turn, causes an elevation in hematocrit without any alterations in RBC concentration or plasma volume, even in refrigerated samples . While the elevations in hematocrit and MCV hinder our analysis of the in-flight data, the stability of RBC count, hemoglobin, MCH, and platelet concentration over 48 h indicates that the observed alterations in these parameters are likely caused by factors associated with space-flight, and are not the result of delayed sample processing.