The hematology and immunology program conducted in support of the Apollo missions was designed to acquire specific laboratory data relative to the assessment of crew health prior to their commitment to space flight. A second, equally important objective was to detect and identify any alterations in the normal functions of the immunohematologic systems attributable to space flight exposure, and to evaluate the significance of these changes relative to man's continuing participation in space flight missions.
Peripheral blood samples were collected by venipuncture L-45 (45 days preflight), L-30, L-15 and L-5, R+0 (on recovery day) and R+1 (1 day postflight), R+6, R+13 and R+16. Radioisotope studies were performed on L-15, R+0, R+6 and R+13.
Hematology analyses ranged from routine procedures intended to provide basic information for the crew surgeon, to more specialized tests designed to elucidate the effects of space flight on the normal functioning and integrity of the red blood cell. For the most part, analyses were performed using standard laboratory techniques.
Determinations of red cell mass and plasma volume were made by obtaining a blood sample and adding iodine 125 (I 125) serum albumin. One portion of this blood was used as a correction factor for circulating radioactivity, another portion was reinjected into the crewmember and the remainder was added to an anticoagulant acid citrate dextrose solution. The chromium 51 (Cr51) isotope was then added to this solution and after 10 minutes ascorbic acid was injected to terminate the tagging process. Another blood sample was drawn for a 15- minute plasma volume specimen, the Cr51 tagged blood was reinjected into the crewmember and the remaining blood was retained to prepare a standard. After 15 minutes, another sample was drawn for a 30- minute plasma volume specimen and a 15- minute red cell mass specimen. Duplicate microhematocrit values were determined on all blood samples. Duplicate 1 ml aliquots of whole blood were pipetted into counting tubes from the initial blood sample, Cr51 standard and red cell mass specimen. All samples were centrifuged and duplicate 1 ml aliquots of plasma were pipetted from each. Triplicate 1 ml aliquots of the I 125 standard were pipetted for counting. Changes in the total amount of circulating Cr51 red blood cells were used to estimate red blood cell survival.
Humoral Immune System
Serum proteins were assayed serially from samples collected on L-30, L-15, L-5, R+0, R+6 and R+16. Serum protein electrophoretic patterns were obtained by cellulose acetate electrophoresis, from which albumin, alpha-2-globulin and gamma-globulin fractions were computed. Individual serum proteins were quantitated by radical immunodiffusion (RID), using specific antisera. Protein assayed by RID include immunoglobins G, A, and M (IgG, IgA and IgM), the third component of complement (C3); the carrier proteins transferrin, haptoglobin and ceruloplasmin; the antiproteases, alpha-1-antitrypsin, alpha-2-macroglobulin, and alpha1-acid glycoprotein.
Cellular Immune System
Lymphocytes were separated from heparinized blood by a nylon reticulum column and cultured with and without phytohemagglutinin (PHA) in appropriate media. At the times of maximal RNA and DNA synthesis, 24 and 72 hours respectively, cultures were pulsed for 1 hour with either 3H-uridine or 3H-thymidine. The incorporation of radioactivity into the washed lymphocytes was measured by liquid scintillation spectrometry. Lymphocyte viability at the time of harvest was assessed by supravital fluorescent staining. The results were calculated as 3H-disintegrations per minute per million viable cells.
Heparinized blood was centrifuged and the buffy coat preserved for chromosome cultures. The cultures were harvested after 66 hours incubation at 37° C. Slides were prepared by the air-dry method and the cells stained with Giemsa -- 200 to 1000 metaphase cells were scored for each individual.
There were no changes in red blood cell count or hematocrit following the flights. Hemoglobin increased slightly from preflight to R+0, resulting in increased calculated mean corpuscular volume, mean corpuscular hemoglobin and mean corpuscular hemoglobin concentration. Platelet counts were normal. Reticulocyte count was slightly decreased on R+0 which is relative to changes in plasma volume and red cell mass.
White blood cell count increased from 7.0 ± 1.8 109 L preflight to 8.9 ± 3.0 109 L at R+0. Lymphocytes slightly decreased 2.6 ± 0.7 109 L preflight to 2.3 ± 1.3 109 L at R+0. The most notable increase was in neutrophil count, 3.9 ± 1.1 109 L preflight to 6.2 ± 2.6 109 L at R+0. As in the Gemini Program, there was a postflight (R+0) leukocytosis associated with an absolute neutrophilia and an absolute lymphopenia. These changes in the white blood cell count and differential were transient and reverted to normal within 24 to 48 hours postflight. While these changes were variable among individuals, they were possibly a consequence of increased blood epinephrine and steroid levels associated with mission stress.
Red cell mass measurement was of particular interest in the early Apollo flights because of significant red cell mass decreases (13 to 21%) observed in the Gemini program. During Apollo 7 and 8, a negligible 2% loss was observed. Apollo 9 showed a 7% decrease. No measurements were made on Apollo 10 through 13 due to constraints imposed by quarantine requirements. Significant decreases (10 ± 1 %) occurred in the Apollo 14-17 lunar landing missions. The deviation in Apollo 7 and 8 from the Gemini crewmen was attributed to a change in the spacecraft atmosphere at launch (100% oxygen in Gemini to a 60% oxygen 40% nitrogen mixture in Apollo). During the Apollo 9 mission, crewmen performed extravehicular activity (EVA) in which denitrogenation occurred after repressurization of the spacecraft and the crew lived in 100% oxygen for the remaining flight. The Apollo 14-17 lunar missions were similar to Apollo 9 in that the Lunar Module purged the Command Module's atmosphere of nitrogen after which the atmosphere was 100% oxygen. The hypothesis from this data indicated hyperoxia (even at low atmospheric pressures) could induce red cell mass loss by hemolysis and/or inhibited erythropoiesis.
The lack of any change in the Cr51 survival time suggests the red cell mass loss may not be due to hemolysis, but to inhibited erythropoiesis. Regardless of the exact cause, compensatory erythropoiesis is not evident. The exact mechanism of red cell mass loss was not established; however, oxygen undoubtedly was a contributory agent, but probably not the only one.
On the missions where red cell membrane lipid peroxides were assayed (Apollo 7, 8, 9, 16 and 17), none were detected. The lack of detectable lipid peroxidation implied that the possibility of overt red cell damage was unlikely.
Changes in plasma volume were more variable, but with a general tendency of being reduced following the Apollo flights, probably due to body fluid shifts in zero-gravity. The rapidity with which the plasma volume can equilibrate makes these results somewhat less meaningful relative to the inflight condition.
The energy-related enzymes showed a postflight elevation, but adenosine triphosphate (ATP) levels were unchanged. The stability of red cell 2,3-diphosphoglycerate is indicative of the maintenance of normal hemoglobin-oxygen affinity. The ATP decline during this study resulted in a significant reduction in the active component of potassium influx in the erythrocytes. However, the decline in potassium influx noted in the Apollo 9 samples was not accompanied by an ATP reduction.
Although some moderate changes were observed during Apollo in the glycolytic enzymes, no trend was evident and the magnitude of these changes did not represent a significant alteration in the functional capacity of the cells.
Methemoglobin concentrations in blood samples collected immediately postflight after Apollo missions 7, 8 and 16 were substantially elevated and remained high in subsequent postflight samples. The magnitude of this elevation was too great to be characteristic of the in vivo situation, and therefore must be assumed to have occurred in vitro during sample storage. It is perhaps significant that the level of methemoglobin in the crew samples was substantially greater than in control samples collected at the same time, and the conversion rate would have to be many times in excess of that reported for stored blood samples. The significance of this finding is unknown, but could have been related to the reverse capacity of the reductase mechanism in the cells in vitro.
In Apollo 13-17, the erythrocytes were examined individually by electron probe microanalysis for cellular sodium (Na), potassium (K), and sulfur (S) content. This consisted of focusing a beam of high energy electrons onto a single red cell and determining the Na, K, and S content by detection of the resultant characteristic X-ray photons emitted from the red cell structural matrix. These procedures were designed to evaluate changes in the population distribution of the two major osmotically active cations (Na and K) of the red blood cell.
The results of these studies on Apollo 13 and 14 showed a transient postflight shift in the red cell Na/K ratios, reflecting an elevated cellular Na content and/or reduced cellular K. This change was not detected on Apollo 15. A significant transient drop in red blood cell K occurred postflight in all three crewmen of Apollo 16. K levels returned to preflight values within one day following recovery. A reduction in erythrocyte S (representative of cellular hemoglobin content) occurred at R+0 on this mission and continued throughout R+1 in samples obtained from one of the three crewmen. No change was observed in the other two crewmen.
Because of the physiological importance of the red cell shape and the variety of pathological conditions in which red cells can undergo shape changes, a characterization system for red cells using the scanning electron microscope was established during the latter Apollo missions to evaluate changes in red blood cell morphology. Six categories of red cell morphology were defined from examination of normal human red cell preparations; these cell types include discocyte, leptocyte, codocyte, stomatocyte, knizocyte and echinocyte. No significant abnormalities were found as a result of space flight exposure to Apollo. However, extensive examination of red cell shape changes in other studies has indicated that red cell morphological alterations do occur during space flight. With only preflight and postflight data from the Apollo missions, any inflight changes might not be detected.
Examination of postflight crew and control group buffy coat (leukocyte) samples by electron microscopy indicated no noticeable changes in intracellular ultrastructure as compared with preflight samples. Mitochondria were intact, cytoplasmic granules were well preserved, nuclear membranes were intact and cell membranes appeared normal.
Humoral Immune System
The total serum proteins were typically increased immediately postflight, with the alpha-2-globulin fraction responsible for this change. The mean concentrations of albumin and the gamma-globulin fractions remained unchanged postflight. The immunoglobulin profiles showed a varied response to space flight. Serum levels of IgG and IgM were unchanged, though individual crewmen demonstrated values in the low normal range but these were consistent throughout the mission. Serum IgA, which includes immunoglobulins responsible for antibiotic, antibacterial, antiviral and isoagglutinin activities, was significantly increased in about one-half of the crewmen on R+0, with a return to preflight levels within a few days.
No significant changes were observed in alpha2-antitrypsin levels, but there was variation among crewmen. The alpha-2-macroglobulin significantly increased at R+0 followed by a rapid drop until rather low levels were obtained by R+16. Concentrations of alpha-1-glycoprotein were unchanged at R+0, but tended to rise during postflight periods.
The increased postflight alpha-2-globulin fraction was a result of hyper-alpha-2-macroglobulinemia and hyperhaptoglobinemia. The consistent postflight elevation of the alpha-2-macroglobulin was puzzling. In the clinical setting, such a change would suggest an underlying nephrotic syndrome; however, in the Apollo crewmen there was no evidence for this disorder. The causes of hyperhaptoglobinemia postflight were elusive. Clearly the etiopathogenic aspects of these alterations in serum levels following space flight warranted further investigation.
Of the three carrier proteins assayed, transferrin, haptoglobin and ceruloplasmin, haptoglobin showed the most consistent and significant postflight change. The mean increase in haptoglobin concentration on R+0 was almost double the preflight levels and was generally still elevated on R+14. A postflight increase in ceruloplasmin was also observed, but it was not as consistent, nor was it as significant as the change in haptoglobin. Transferrin showed no significant change immediately postflight but tended to decrease during the two-weeks postflight.
The astronauts as a group demonstrated certain characteristic serum profiles following exposure to the space environment: elevated alpha-2-globulin due to increases of haptoglobin, ceruloplasmin and alpha-2-macroglobulin; elevated IgA and C3 without evidence of compromised humoral immunity; and a delayed postflight depression of alpha-2-macroglobulin, which was transiently elevated after splashdown and recovery. Transferrin tended to decrease at the end of the second postflight week. The mechanisms responsible for these changes are unknown.
While these patterns prevailed for the crewmen as a group, individual crewmen demonstrated interesting exaggerations or mitigations of these mean changes. One crewman exhibited a marked increase of acute phase reactants such as haptoglobin and alpha-1-antitrypsin, with a depression of transferrin levels. This pattern contrasted with that of the other crewmen in the group, for which changes in alpha-1-antitrypsin were insignificant. This crewman had experienced a pyelonephritis secondary to a Pseudomonas infection. Another crewman experienced an episode of mild otitis media which was coincident with a decrease of IgG to approximately two-thirds of normal levels. The possibility cannot be excluded that this reduced IgG level may have contributed to this individual's susceptibility to infection.
There were no consistent abnormalities relative to the humoral immune system due to space flight. There were unexplainable characteristic alterations in some of the proteins, haptoglobin and alpha-2-macroglobulin in particular. However, the medical consequences of these changes relative to man's immune competence during and after space flight would appear to be minimal. There were no indications from these data to suggest that the functional capacity of the immune system is restrictive to man's participation in space flight.
Normal in vitro lymphocyte synthesis of nucleic acids, in both the basal unstimulated state and in response to the stimulating agent PHA, tended to remain well confined within relatively narrow ranges of variability, irrespective of the lymphocyte counts of each individual. The RNA and DNA synthesis rates for lymphocytes cultured before and after flight from crewmen from Apollo 7 through 13, remained well within the normal 90% range. In Apollo 14-17, the data were somewhat less consistent but were within normal ranges, both preflight and postflight, and therefore fit the general trend.
Cellular Immune System
Evaluation of the cellular immune response of lymphocytes from preflight and postflight blood samples of the Apollo 16 crew strongly suggested the presence of a subclinical viral infection in both the prime and backup crew. These indications were based on abnormal rates of RNA and DNA synthesis in unstimulated lymphocytes as indicated by radioactivity count levels above and below the normal ranges, and abnormal high or low values for rates of RNA and DNA synthesis in PHA stimulated lymphocytes. Electron microscope studies of lymphocytes from the prime crew at R+3 provide a supplemental evidence of subclinical viral infection, based on increased protein-synthesizing capacity. These conclusions were supported by preflight and postflight incidences of lymphocytosis and a high percentage of atypical lymphocytes.
As in the Gemini Program, there was a postflight (R+0) leukocytosis associated with an absolute neutrophilia and an absolute lymphopenia. White blood cell count increased (7.0 ± 1.8 109 L preflight to 8.9 ± 3.0 109 L at R+0). The most notable increase was in neutrophil count (3.9 ± 1.1 109 L preflight to 6.2 ± 2.6 109 L at R+0). Lymphocytes slightly decreased (2.6 ± 0.7 109 L preflight to 2.3 ± 1.3 109 L at R+0). Changes in white blood cell count and differential were transient and reverted to normal within 24 to 48 hours postflight; they were possibly a consequence of increased blood epinephrine and steroid levels associated with mission stress.
While individual crewmembers exhibited a variability in lymphocyte patterns preflight and postflight, the majority exhibited a significant but fluctuating increase in lymphocyte numbers shortly after, but not coincident with recovery. The mean lymphocyte count for all Apollo crewmembers reflected a value which that remained within the normal range. Based on a normal human peripheral blood lymphocyte mean count of 2400 per cubic millimeter and a range of approximately 1500 to 4000 per cubic millimeter; 20 of the 33 crewmen exhibited early postflight increases above the normal mean, and five of the 33 above the upper limit of the normal range. Five crewmembers experienced lymphocyte counts below the normal range.
The significance of the lymphocyte pattern is unknown. Several factors must be considered in the context of the normal environment during space flight. Among these are demargination and mobilization of lymphocytes from sequestered pools, adrenal corticosteroid influences, possible effects of radiation and impaired recirculation pathways.
The ability of small lymphocytes to respond to antigenic stimulation by PHA with increased synthesis of RNA and DNA was associated with characteristic morphological changes, and is generally accepted as an in vitro indicator of in vivo immunocompetence of T-cells. These morphologic alterations are paralleled by functional changes, such as increased RNA and increased DNA synthesis rates.
The rates of spontaneous unstimulated and PHA-stimulated synthesis of both RNA and DNA by lymphocytes cultured preflight and postflight remained within the 90% normal range with the exception of the Apollo 15 and 16 crews.
While lymphocyte numbers fluctuated significantly shortly after return from space, and tended to exhibit a delayed increase, the immunocompetence of these cells, as judged by in vitro stimulation techniques, remained stable throughout the preflight and postflight observations. This finding is of significance in engendering confidence that the immune system, particularly such vulnerable components as circulating antigen-sensitive small lymphocytes, can maintain functional integrity in the space environment. The influence of longer duration flights may be more complicated and could influence the lymphocyte responsiveness postflight.
Chi-square tests on preflight versus postflight aberration rates showed that approximately 50% of the crewmen tested had significant increases in chromatid-type changes postflight. Fewer tests showed significant chromosome-type increases. If the Apollo crewmen are divided into two groups based on the presence or lack of previous flight experience, an interesting fact emerges. Only one out of six crewmen who were on their first mission had a preflight value above four percent, whereas all but one of the nine experienced crewmen had preflight values of four percent or more.
The postflight break rates were frequently higher for Apollo than for Gemini, and the overall means were nearly double (7.73% versus 3.94%). With the longer duration Apollo missions there was a corresponding increase in postflight aberration yields. Although there was wide variation in individual values, the trend is apparent.
Chromosome analysis suggested postflight chromosome aberrations were approximately double preflight values. There was a rather constant postflight aberration yield which seemed to be dependent of the duration of the flight, and baseline or preflight values in experienced astronauts appeared to be higher than in the other crewmen.
Although there were subtle alterations for some aspects of erythrocyte function, plasma protein profiles, lymphocyte response patterns, and chromosome aberrations, none of these changes compromise man's performance capacity while in space or should limit his stay in space. While questions remain unanswered, especially with respect to longer duration missions, no drastic alterations were observed during the Apollo program for the hematological and immunological systems which would cause serious concern for the health and safety of the crewmen on longer space journeys.
|Mission||Launch/Start Date||Landing/End Date||Duration|
|Apollo 10||05/18/1969||05/26/1969||8 days|
|Apollo 11||07/16/1969||07/24/1969||8 days|
|Apollo 12||11/14/1969||11/24/1969||10 days|
|Apollo 13||04/11/1970||04/17/1970||6 days|
|Apollo 14||01/31/1971||02/09/1971||9 days|
|Apollo 15||07/26/1971||08/07/1971||12 days|
|Apollo 16||04/16/1972||04/27/1972||11 days|
|Apollo 17||12/07/1972||12/19/1972||12 days|
|Apollo 7||10/11/1968||10/22/1968||11 days|
|Apollo 8||12/21/1968||12/27/1968||6 days|
|Apollo 9||03/03/1969||03/13/1969||10 days|