Hematology and Immunology Studies

A COORDINATED SERIES OF EXPERIMENTS (The M110 Experiment Series) were conducted in support of the Skylab Program for the primary purpose of evaluating the specific aspects of immunologic and hematologic system responses of man to, or alteration by, the space flight environment. Particular results of two of these experiment protocols, "M112-Man’s Immunity, in vitro Aspects" and "M115-Special Hematologic Effects," are the subject of this chapter. The selection of tests for these experiments and others of the M110 Series was biased by the results of medical studies conducted in support of the Gemini and Apollo flight programs. Data from these missions suggested possible influences of the space flight environment on red cell integrity and the normal regulation of the circulating red cell mass. The concept of rapid shifts in body fluid compartments during the transition between a normal one-g environment and the weightless condition also represented stresses of unknown magnitude to the body’s homeostatic mechanisms. Results of immunological studies from the Apollo Program indicated that an acute-phase protein response might be characteristic of space flight. There was also concern relative to the immune competence following an extended period of time in the relatively closed environment of the Skylab orbiting workshop.

These aspects, plus the opportunity to investigate man’s biochemical response to an extended exposure to the weightless environment of space flight, provided the scientific background and impetus for the formulation of these medical studies. Blood samples were collected by venipuncture from each of the three Skylab crews and from ground-based control subjects periodically during the preflight, in-flight, and postflight phases of each mission. The backup crews for each mission were also studied during the preflight period. Depending upon the assay to be conducted, different anticoagulants were used; however, all samples were processed or stabilized within minutes of collection. In-flight samples were collected in Na2EDTA and immediately separated by centrifugation into plasma and cellular phases by a device especially designed for operation in a weightless environment (appendix A, sec. I.e.2). Following separation, the in-flight samples were frozen at -20 ° C and stored onboard until recovery, whereby the specimens were then transferred to the laboratory for analysis. The total volumes of blood collected during each phase of a mission are summarized in table 28-I.

Immunology Studies

The assessment of man’s immunologic integrity was of particular importance in evaluating the medical consequences of manned space flight. The Skylab study was undertaken to monitor specific plasma proteins (table 28-II) prior to, during, and immediately following the extended space flight exposure. The primary objectives of this investigation were:

To assess the status of crew health prior to launch;

To establish individual baseline data for later comparisons; and

To detect possible aberrations of the immune system as a result of exposure to space flight, particularly with respect to its capacity to respond after a lengthy time in the relatively closed environment of the Skylab Workshop.

Prior to the Skylab flights, the question of compromise to the immune system from the lack of exposure to multivarient challenges was considered a potential problem.

Certain alterations in serum proteins as a result of exposure to space flight were a consistent feature of the immunology studies during the Apollo Program (refs. 1,2). These consisted of a significant rise and subsequent decrease of alpha2-macroglobulin and a rise in haptoglobin levels postflight. Moderate increases in Complement Factor 3 (C3), ceruloplasmin and alpha1-acid glycoprotein were also observed after some missions.

Analysis of proteins during Skylab were performed with EDTA-collected plasma instead of serum. This change was necessary so that preflight and postflight samples would be comparable to those collected during the inflight phase of the mission. A list of the plasma proteins analyzed during the Skylab flights is detailed in table 28-II.

Plasma protein profiles after Skylab 2 the first manned Skylab Mission (28 days) may be summarized as follows:

Total Proteins and Electrophoretic Patterns.No significant changes were observed.

Immunoglobulins. No significant changes were observed, although the Pilot had high IgA levels throughout the study.

                     Protease Inhibitors. No significant changes were observed.

Complement Factors. There was a slight decrease in C3 immediately postflight in all three crewmen. By 13 days postflight all values were within the preflight normal levels.

Other Proteins. The Scientist Pilot and Pilot had increased levels of lysozyme postflight that were still elevated by 13 days postflight.

The protein aberrations noted during the Apollo Program were not evident in this mission nor the two subsequent manned Skylab flights (Skylab 3 and Skylab 4); in addition, there were no significant modifications in any of the plasma proteins during the 59-day Skylab 3 or 84-day Skylab 4 missions. Thus, there were no indications of a response of the humoral immune system to the conditions of weightless flight characteristic of Skylab nor of changes in the system’s capacity to respond to a foreign challenge. It can only be speculated that the increased amount of physical exercise during Skylab 3 and particularly Skylab 4 may have resulted in a prevention of these alterations of serum protein profiles, in spite of the extended time periods of these missions.

In the Skylab 2 astronauts, the observed slight decrease of C3 and increase of serum lysozyme levels cannot be fully explained at present. These changes may, however, also be related to the a2-macroglobulin changes, since a secondary relationship may exist. Recruitment of the classical complement sequence by plasmin activation of C1 to C3 (ref. 3) leads to the initiation of the terminal complement amplification mechanism by plasmin cleavage of C3 (ref. 4) and release of lysosomal enzymes from polymorphonuclear leukocytes. Goldstein, et al., (ref. 5) have demonstrated that complement activated via the alternate pathway interacts with human polymorphonuclear leukocytes (PMN) in the absence of particulates and stimulates the selective release of lysosomal enzymes. "Levels of C3 proactivate . . . correlated inversely with and perturbs the PMN plasma membranes sufficiently to cause lysosomal membrane perturbation, fusion, and ultimately lysosomal enzyme extrusion by a process of reverse endocytosis."

The functional capacity of the cellular immune system was evaluated based upon the ability of purified lymphocyte cultures to undergo blastoid transformation in response to an in vitro mitogenic challenge. The agent used was phytohemagglutinin (PHA). The ribonucleic acid (RNA) synthesis rate after 24 hours in culture and the deoxyribonucleic acid (DNA) synthesis rate after 72 hours were determined from the uptake of 3H-uridine and 3H-thymidine, respectively. Techniques utilized for these studies on Skylab astronauts and their controls were exactly the same as those used for the studies on the Apollo astronauts and the SMEAT control persons, except for one aspect: 10 percent normal human AB serum in TC-199 was employed instead of the 10 percent fetal calf serum in TC-199, since homologous AB serum, in general, eliminates possible nonspecific stimulation of human lymphocytes by calf serum proteins. Five percent of CO2 in air was used to maintain pH values of the culture media throughout the culture periods. In addition to studies of the in vitro lymphocyte responsiveness to PHA, two more tests were added for the evaluation of thymus-dependent lymphocyte (T-cell) functions, i.e., mixed lymphocyte cultures using a technique described by Bach and Voynow (ref. 6) and rosette formation of lymphocytes with sheep erythrocytes (E- or T-rosette).

The results of these lymphocyte studies are shown in figures 28-1a, 28-b,28- c; 28-2a,28-2 b,28-2c. These results indicate that the PHA responsiveness of lymphocytes remained normal under a ground based simulation of the Skylab environment (SMEAT), but decreased markedly in most Skylab astronauts on the day of recovery. This finding, characteristic of all three missions, did not appear to be related to the duration of the flight. The capacity of the lymphocytes to respond to PHA was recovered rapidly, and by 3 to 7 days postflight was within the established preflight limits.

Because of these changes, additional studies were conducted on the Skylab 4 mission to measure the response of the cells in a mixed lymphocyte culture and to quantitate the B- and T-lymphocyte distribution preflight and postflight. In contrast to the changes in PHA responsiveness, the Skylab 4 crew’s lymphocytes showed normal mixed lymphocyte culture response patterns on the day of recovery. Thus, although one index of T-cell function, PHA-responsiveness, decreased temporarily after space flight, another measure of T-cell function, the mixed lymphocyte culture response, showed no significant change. There was a reduction in the number of circulating T-cells on recovery day, as determined by the E-rosette procedure, with a return to normal levels by day 3 postflight. Interpretation of the response of the cellular immune system to space flight is further complicated by a transient elevation in the number of B-cells on day 1 postflight, with a rapid return to normal levels by day 3 postflight.

Lymphocytes from the crew of Skylab 4 were also examined by scanning electron microscopy, and classified relative to the density and length of the surface microvilli. It has been recently proposed that B- and T-lymphocytes could be identified by the degree of microvilli present on their surface when fixed for electron microscopy (ref. 7). Examples of the differences in lymphocyte surface features may be seen in figures 28-3a, 28-3b,28-3 c. More recently, the classification of lymphocytes as B- or T-cells by this method has not withstood the test of time and further investigation. It is probable that the density and extent of the microvillious network is related to the state of activity of the cell at the time of fixation. Thus smooth T-cells develop extensive microvilli when exposed to sheep erythrocytes during the E-rosette procedure (ref. 8), and these microvilli might be the points of attachment of the red cells to the lymphocyte (ref. 9) . At any rate, examination of thousands of lymphocytes from the crew preflight and postflight indicated no significant difference in the percentages of smooth, intermediate, or hairy cells.

The effects of certain endogenous factors, such as corticosteroids and catecholamines, upon lymphocyte functions require elucidation. It has been shown that the intravenous injection of large dosages of hydrocortisone in healthy adults results in decreased in vitro lymphocyte responses to various mitogens (ref. 10). It has been shown that steroids (e.g., prednisone) depress both the circulating B- and T-lymphocytes numerically and functionally. Depressed functions of B-lymphocytes are reflected by a fall in serum immunoglobulin levels, and those of T-lymphocytes, by a reduced PHA-response. The effects of corticosteroids are rapidly expressed and abate equally rapidly with cessation of the drug.

In contrast to corticosteroids, adrenal medullary hormones, such as epinephrine and norepinephrine, chemically defined as catecholamines, stimulate adenyl cyclase activity in murine (ref. 11) and human lymphocytes (ref. 12). Such stimulatory effects of catecholamines are probably ß-adrenergic in nature (ref. 11). Since the elevation of adenyl cyclase activity, followed by increased levels of cyclic adenosine monophosphate, is one of the earliest biochemical changes in lymphocytes following stimulation by mitogens and antigens (ref. 13), these ß adrenergic effects of adrenal medullary hormones may affect both the in vivo and in vitro functions of lymphocytes, and may have direct bearing on the mechanisms of temporarily altered lymphocyte functions associated with manned space flights.

The absolute white cell count was typically elevated at recovery (figs. 28-4a, 28-4b,28-4 c; tables 28-III, 28-IV, 28-V), but rapidly returned to preflight levels. Differential counts indicated that this elevation was due to an absolute increase in the number of neutrophils with the lymphocyte absolute count not changing significantly. These results are consistent with the high cortisol levels measured in the crews at recovery (ch. 23).

The medical significance of these changes in the cellular immune system is not clear at this time. It is difficult to predict what a reduced PHA responsiveness, associated with a reduced number of T-cells, of this magnitude and short duration means with respect to the immune competence of the returning crews. The Skylab crews were maintained in isolation for 7 days postflight; thus, their potential for contact with infectious agents during that time was significantly reduced. The exact cause and impact of the reduced lymphocyte responsiveness needs clarification for planning the postflight activities of future space flight crews.

Hematology Studies

Measurements of hemoconcentration parameters, red blood cell count, hemoglobin concentration, and hematocrit, were complicated during the postflight period by rapid changes in plasma volume and more gradual recovery of the reduced red cell mass. There were also differences between Skylab 2 and the two longer missions, presumably because of the varying rates of recovery of the phases of the vascular volume.

The red cell count, hemoglobin concentration and hematocrit were below preflight levels in all three crewmen immediately following the Skylab 2 flights. The red cell count showed a gradual recovery by days 4 to 7 postflight, but the hematocrit and hemoglobin concentration were below preflight levels during the 18-day postflight period (figs. 28-5a, 28-5b,28-5c)  The mean corpuscular volume (MCV) showed an elevation on recovery and the day after recovery as would be predicted based upon the red cell count and hematocrit data for those examinations. By day 4 postflight the MCV was slightly reduced in all three crewmen below the preflight mean. However, this depression was not statistically significant. The mean corpuscular hemoglobin (MCH) and mean corpuscular hemoglobin concentration were slightly elevated on recovery and the day after recovery day tests but showed no significant change from preflight baseline levels during the postflight period.

The 59-day and 84-day flights were similar with respect to the response of the red cell count and associated measurements, and both were somewhat different than the data from Skylab 2. The red cell count, hemoglobin concentration, and hematocrit were elevated on recovery as compared to preflight levels (figs. 28-6a, 28-6b,28-6c), but began to decline by day 1 postflight and by day 3 postflight were significantly lower than preflight values. In both missions, these hemoconcentration-dependent parameters gradually returned to within preflight limits during the 3-week postflight examination period. Red cell indicies (MCV, MCH, and MCHC) showed considerable variation postflight, but none significantly out of the established pre-flight limits.

The differences and similarities between the three missions with respect to changes in the fluid and cellular compartments during the immediate postflight period are evident from the data on hemoglobin (table 28-VI). The hemoglobin concentration following Skylab 2 was decreased slightly (-6.1 percent) on recovery and changed very little by day 3 postflight (-7.5 percent) com-pared to the preflight values. The hemoglobin concentration in the crews of Skylab 3 and Skylab 4 were, in contrast, elevated on recovery (4.2 per-cent). By day 3 postflight the values had declined by 15.4 percent compared to recovery and 11.9 percent relative to the preflight mean. These results are most likely due to plasma volume shifts, but may have a bearing on the recovery kinetics of the red cell mass during the immediate post-flight period.

If the red cell mass loss data during the three missions are considered as a composite, a time-relationship between the red cell mass change (in percent) and days following launch becomes apparent (fig. 28-7). These results suggest that following some initial insult during the first 2 or 3 weeks of flight, the red cell mass begins to recover, after a refractory period, at about day 60. An interesting point from these data is that the initiation of recovery would appear to be independent of the presence, or absence, of gravity. The 2-week period of no recovery following Skylab 2 could, in part, be related to the somewhat normal concentrations of circulating hemoglobin during that time. The crews of Skylab 3 and Skylab 4, which began to make up their red cell mass deficit almost immediately, had hemoglobin concentration values, during this time period, that were lower than their preflight mean levels.

Additional data from in-flight hemoglobin measurements would lend support to this hypothesis. Hemoglobin concentrations were measured in-flight using a hemoglobinometer in conjunction with each in-flight blood draw on the second and third mission. Duplicate measurements of the hemoglobin concentration in the hemolysate of each of the returned frozen in-flight blood samples were made postflight using the cyamethemoglobin procedure. The whole blood hemoglobin concentrations in these samples were calculated using the relative volumes of the plasma and cellular compartments and the following equation:

HB = HB(READ) X HVOL/(HVOL+PVOL)

where HVOL is the volume of the hemolysate returned in the automatic sample processor (ASP). PVOL is the volume of plasma in the plasma cartridge (PC) and HB (READ) is the hemoglobin concentration of the hemolysate. HVOL and PVOL were calculated from gravimetric measurements of the ASP and PC contents. The specific gravity of the hemolysate was assumed to be 1.09 and that of the plasma 1.03.

Because of the conditions under which the in-flight samples were assayed and the procedure used, and because of the calculations and assumptions required to determine the hemoglobin concentrations in the returned samples, the precision of the values associated with the in-flight blood samples is less than that of preflight and postflight determinations.

The ground-based laboratory measurements were generally lower than in-flight determinations, particularly on the second mission (figs. 28-8a,28-8b,28-8c; 28-9a,28-9b,28-9c).

The reason for this difference is not known at this time. Postflight comparisons of the Skylab 3 Scientist Pilot’s hemoglobin readings using the flight hemoglobinometer with laboratory measurements of the same blood samples (crew and standards) at day 3 postflight indicate no significant difference. There was relative agreement between plasma volume measurements and the trend of the hemoglobin concentrations in-flight. Consistent in all the in-flight hemoglobin determinations was an elevated value in the first in-flight sample, presumably due to a loss of plasma volume, and a gradual reduction in the hemoglobin concentrations as the mission progressed.

If the relative hemoglobin concentrations, calculated as percent of the preflight mean and averaged for each crew, are examined, a downward trend with time is evident (fig. 28-10). It is perhaps significant to note that the in-flight hemoglobin level drops below the preflight mean (100 percent) only after day 60, about the same time that the recovery of the red cell mass is initiated (fig. 28-7). Thus, if the concentration of hemoglobin is a significant factor in stimulating erytnropoiesis, it is perhaps significant that during the first 60 days in-flight the hemoglobin is above its preflight level. This concept could then explain the delayed recovery of red cell mass in the Skylab 2 crew after recovery. These data are not conclusive evidence of the mechanism by which the erythropoietic processes fail to compensate for reduced levels of circulating red cells, but do offer possibilities for a contributing factor, and do provide information relative to plasma volume shifts during and immediately following exposure to weightlessness. The kidneys use both changes in hemoglobin concentration and oxygen delivery to modulate erythropoietin release. Thus, the decreased red cell masses of the Skylab crewmembers might not be followed by compensatory increases in erythropoietin until the plasma volume increased. Without increased erythropoietin, bone marrow activity would not increase and would appear inhibited until a new equilibrium was reached.

Red cells were examined for changes in their specific gravity profile using the procedure described by Danon and Marikovsky (ref. 14). A series of phthalate esters of varying concentrations were utilized to separate each red cell popuation into a density profile containing 13 fractions with specific gravities ranging from 1.057 to 1.136. There was considerable difference between individual crewmen but little variation for an individual subject. A consistent shift in the specific gravity profile immediately postflight toward a population of heavier cells was observed. The most dramatic changes were observed after the shorter Skylab 2 flight (figs. 28-11a, 28-11b,28-11c). In all missions the changes were transient and by day 3 postflight, population distribution of the cells were within preflight ranges. The possible reasons for these shifts in specific gravity distribution in the red cell population have been discussed previously (ref. 15) but the actual cause cannot be established at this time. However, because of the rapid reversal in distribution to within normal limits it seems likely that the change in specific gravity profile is not indicative of a change in the average red cell age. It would appear more likely that the change represents an alteration of red cell membrane lipid content, cell water content, cell electrolyte concentration, or a combination of the three.

The "active" and "passive" components of potassium (K) influx into red cells were determined using a ouabain inhibition, 86Rb substitution technique (ref. 16). There were no significant changes in either the passive or active components of potassium influx following the first Skylab flight. However, after Skylab 3 and Skylab 4 there were significant elevations in the ouabain sensitive, metabolic-dependent potassium influx in four of the six crewmen. This elevation in the metabolic-dependent component of potassium influx was not excessive (mean=46 percent). The rate of accumulation of potassium in red cells has a measured maximum of about 4 mEq per liter cells per hour. The increase in potassium influx observed at recovery had returned to normal rates by days 3 to 7 postflight. This change may be interpreted as a response to a transient alteration in the dynamic state of equilibrium existing across the cellular membrane with respect to electrolytes and water. A temporary reduction in cell water would make the cell heavier (as observed by their specific gravity profile) and would also stimulate the sodium-potassium (Na-K) "pump." A permanent elevation would be suggestive of membrane damage resulting in a more permeable cell. The osmotic fragility data indicate that the resistance of the red cells to osmotic stress has not been compromised during the mission. The levels of high energy phosphate components (ATP and 2,3-DPG) were also unchanged during the flight (ch. 27).

The potassium content of the red cells (as measured by flame photometry) did not change significantly during the mission in either the light cell fraction (LCF) (younger cells), heavy cell fraction (HCF) (older cells) or unseparated blood samples (UNS) (table 28-VII). The red cell separation technique used was that of Herz and Kaplan (ref. 17).

Red Cell Shape Classification

The familiar biconcave discoid shape of the mature erythrocyte represents a unique structural configuration among cell types. This peculiar shape is so consistent and characteristic of normal erythrocytes that deviations from the discoid form have provided the bases for the detection and diagnosis of a variety of congenital and acquired hemolytic disorders (refs. 18,19,20,21,22,23). The mechanisms involved in the maintenance of this biconcave shape have been of considerable interest to physiologists, chemists, and mathematicians for a number of years. Several theories have been proposed to explain the physical and chemical bases of this configuration (refs. 24,25,26,27,28), but as yet no single explanation is acceptable to all investigators.

Regardless of the exact mechanism by which the red cell maintains its "normal" discoid shape and regardless of the advantages or disadvantages of this shape relative to the red cell functions (i.e., optimum gas exchange, deformability, survival), it is quite evident that a delicate balance exists between the chemical and physical forces and the metabolic energy and ultrastructual organization of molecules—all interacting to exert a complex array of vectorial forces on the red cell membrane. It is probable that alterations in this balance of forces, exhibited by the red cell, are responsible for a variety of different morphological states ranging from a discocyte to a spherocyte with many intermediate shapes. This imbalance may be the result of an intrinsic metabolic or structural defect of the cell usually characterized by a hemolytic anemia.

A second class of factors causing alteration in the red cell shape includes extrinsic properties of the plasma milieu. This second type of shape change is usually of a less severe nature and, provided the cell is not destroyed by selective removal in the reticuloendothelial system (RES) or hemolyzed due to an imbalance of ion and water regulation, these changes are reversible if the causative agent is neutralized or removed from the plasma. The most common and most widely investigated type of red cell shape change due to extrinsic factors is the conversion of the normal discocyte to a spiculed cell, the discocyte-echinocyte transformation. Thus, the evaluation of this type of reversible change in red cell shape may not only provide an indicator of alterations in red cell functional capacity but also may be used to detect and identify subtle changes in plasma constituents (especially those known to have cytogenic properties relative to red cell shape).

As one aspect of the protocol for Skylab Experiment M115, Special Hematologic Effects, samples of blood collected from the crewmen preflight, in-flight and postflight were critically examined by light and scanning electron microscopy for alterations in the shape of the red blood cells. This study was designed specifically to investigate, detect, and characterize alterations in red cell shape either during or following extended exposure to the space environment. The following data will describe the alterations in red cell shape observed during the extended Skylab space flights and the rapid reversal of these changes upon reentry to a normal gravitational environment. Possible causes for these modifications in red cell shape will be discussed, as will the significance of these changes to man’s functional capacity in space and to other observed hematologic events.

Red blood cells from astronaut crews were processed for scanning electron microscopy (SEM) using the following procedures.

Fixation.—Blood samples from preflight and postflight medical examinations were collected in heparin; in-flight samples were taken in EDTA. Approximately 0.1 ml of whole blood per sample was added to 1.0 ml 0.5 percent glutaraldehyde, pH 7.4, 320 mOsm, prepared in a standard incubation medium. Time in the fixative varied from 1 hour for preflight and postflight samples to 1, 2, 24, 57, and 81 days for in-flight samples. No effect was found on cell morphology as a result of the varying lengths of time the red cells spent in glutaraldehyde. The fixed cell samples were washed twice in a standard incubation medium, pH 7.3, 300 mOsm, and then twice in deionized water prior to critical point drying.

Dehydration and Critical Point Drying.—Each red cell sample was allowed to sediment for 5 minutes from water onto a clean 9 X 22 mm glass cover slip without air-drying. The sample was dehydrated to 100 percent EtOH by gently adding graded EtOH solutions dropwise to the water on the cover slip. Three rinses were made with each solution; the third rinse was allowed to remain on the cell sample for 5 minutes prior to replacement with the next solution. A stepwise series of 20, 50, 75, 90, and 100 percent EtOH solutions were used. The EtOH was then replaced with 50 percent amyl acetate/50 percent EtOH and finally 100 percent amyl acetate. The samples were critical point dried from liquid carbon dioxide using a Denton critical point drying apparatus.

Coating.—The glass cover slips with the red cell samples were mounted on aluminum studs using double-edge conductive tape and silver conducting paint. The samples were then coated with approximately 300 anstram gold/palladium (60 percent/40 percent) in an Edwards evaporator equipped with a rotary/tilt stage.

SEM Examination.—The red cell samples were examined in an ETEC Autoscan at 20 kV, with 2000 X magnification. Resolution of the microscope under these conditions is on the order of 200 anstram. Magnification and other instrument parameters were held constant for all red cell classification.

Classification.—A quantitative, differential classification scheme for red cell shapes was developed and tested by the Cellular Analytical Laboratory at JSC, in ground-based studies (SMEAT and ground control subjects), and in support of the Apollo 17 mission, prior to its implementation in the Skylab Program. The criteria for differentiation of cell shapes and the terminology used are outlined in table 28-VIII and are comparable to those recently discussed at a workshop on red cell shape at the Institute of Cell Pathology, Hopital de Bicetre, Paris (ref. 18).

This classification of red cell morphology by shape rather than by disease or origin appears to be desirable from the standpoint that similar or identical shapes may arise from more than one type of disorder or condition. The terminology proposed by Bessis will be used throughout the following discussion.

In each red cell sample, from 500 to 1000 red cells were examined and classified into one of four distinct groups of cells. For the third Skylab mission, this classification scheme was enlarged to include two additional categories. Examples of the types of red cell shapes observed in the Skylab samples are illustrated in figure 28-12,fig. 28-13, fig.28-14,fig.28-15, fig.28-16,  fig.28-17, fig.28-18, fig.28-19, fig.28-20, fig.28-21, and fig. 28-22.

Light Microscopy.—Red blood cell smears were prepared for routine examination using standard hematological procedures with Wright’s stain.

Routine hematologic red cell smears prepared from blood samples collected immediately post-flight (within 2 hours of splashdown) and examined by light microscopy (oil-immersion, 1000 X magnification) were by all standard criteria essentially normal. There were no obvious variations in the size or shape of the cells as compared to pre-flight samples. Cell edges were smooth, and the cells were essentially normochromic with no evidence of cytoplasmic inclusions. Quantitative microspectrophotometric examination of single cells indicated no change in the hemoglobin content, and the calculated MCH and MCHC were also normal. Unfortunately no slides were prepared during the in-flight phase of the missions for comparison.

Quantitative classification of the red cell population, based on variations in cell shape as determined by scanning electron microscopy, indicates a significant variation in the distribution of cell types during the in-flight portions of each mission (fig. 28-23 through 28-25). During the preflight phase 80 to 90 percent of the circulating red cells were classified as discocytes (mean=83.4 ±10.3), but there was considerable variation among individual crewmembers (range, 60.9 to 92.9 per-cent). The percentage of discocytes in the blood samples collected immediately postflight (mean= 82.7  = or -7.9) was not significantly different from preflight levels among the crews as a group or among individuals. The remaining 15-20 percent of the nondiscoid cells present during the preflight control phase of each mission consisted primarily of leptocytes, stomatocytes, and knizocytes (see fig. 28-14, fig 28-15, fig.28-16, fig.28-17, and  fig.28-18) with the frequency of echinocytes (fig. 28-19, fig.28-20) present being less than 1 percent.

During exposure of the crews to the space flight environment, the frequency of echinocytes increased significantly, and this increase appeared to be related to the duration of each mission (fig. 28-26). Again, considerable individual variation was evident (figs. 28-27, fig.28-28, and fig. 28-29) but the increase in the numbers of echinocytes, expressed as an average of each crew, was statistically significant after the first sampling period of each mission. The majority of the echinocytes present in these samples were of the stage I type (figs. 28-19, 28-20), with few progressing to stages II or III (figs. 28-21, fig.28-22). The first sample collected postflight (Recovery + 0 days, R+0) was prepared within 2 hours of reentry of the spacecraft. The number of echinocytes observed in this sample represented less than 1 percent of the red cell population and was, therefore, comparable to the preflight value. This rapid reversal of the discocyte-echinocyte transformation is significant and will be discussed in detail below.

The pattern of change observed with respect to increases in the numbers of stomatocytes and knizocytes was different from that recorded for transformation to echinocytic shapes. If the data from all three missions are considered as a composite, there appears to be maximum increase prior to mission day 27 (MD27) and a gradual reduction with continued time in-flight (fig. 28-30). The percentage of stomatocytes and knizocytes present on MD82 is not significantly different from that on recovery day (R+0). It is possible that these altered cells underwent a further transformation to an echinocytic type later in the mission. The entire crew and particularly those individuals exhibiting the greatest change in the number of echinocytes (Pilot-3, Commander-4, and Pilot-4) did not show a further reduction in their discocyte frequency after the first in-flight sample. (The response of the Pilot-4 is an exception and will be discussed below.) The mean discocyte frequency in 8 of the 9 crewmen was 82.6% ±10.3% on MD3-4 as compared to 81.0% ±6.8% on the second sampling day (MD27, MD58, or MD82). However, these values may be somewhat misleading because of the individual variation and relatively small sample size.

The kinetics of the transformation from discocyte to leptocyte demonstrated even a third pattern, with only two of the three crewmen of the 84-day mission (Skylab 4) showing a significant elevation in the frequency of this cell type (fig. 28-31). Even among the Skylab 4 crew the increased average frequency is due primarily to the response of the Pilot-4 (fig. 28-32) with the other two crewmen showing only a slight elevation earlier in the mission. It should be noted that the Pilot-4 had a high percentage (15.5) of leptocytes present during the preflight phase and the lowest percentage (60.9) of discocytes of the nine crewmen examined (fig.28-23, fig.28-24,and fig. 28-25).

Attempts to compare the degree of change in red cell shape with alterations in several plasma and cellular constituents (Na, K, Ca, Mg, Cl, osmolality, ATP, 2,3-DPG) failed to demonstrate a significant linear correlation. This finding was not surprising when one considers the sparsity of data values and the inherent weaknesses in the mathematical determination of linear correlation coefficients. Unfortunately data relative to other plasma echinocytogenic factors (especially lecithin and lysolecithin, cholesterol, and free fatty acids) and their cellular concentrations were not available for comparison.

Similar studies were done in support of Apollo 17 and the Skylab Medical Experiments Altitude Test (SMEAT) at JSC. There were no significant changes in red cell shape distributions during the 56-day SMEAT study in the three-man crew (discocyte mean for entire study = 85.0% ± 3.9%) or ground-based control group (mean = 78.9% ± 4.4%) either during or immediately following the exposure period (ref. 29). On Apollo 17 the post-flight percentage of discocytes (84.0% ±6.5%) was not significantly different from preflight crew values (90.4% ± 3.6%) or those of the control group (preflight mean = 87.3% ±11%, postflight mean = 90.5% ± 3.3%). The Skylab ground control group had no changes during the in-flight phase when fixed red cells were maintained exactly as those prepared by the astronauts.

The results of this study suggest that during extended exposure to the space flight environment significant alterations occur in the distribution of red cell shapes in the peripheral circulation. The most consistent change observed was the discocyte-echinocyte transformation which was readily reversible following completion of the mission. The kinetics and causes for this type of red cell shape change have been extensively studied in both in vitro and in vivo systems (refs. 19,20,21,22,23, 30,31,32,33,34,35,36). The concept of echino-cytogenic plasma, plasma capable of crenating normal red cells, has been well documented by these investigators. Various echinocytogenic factors identified thus far are summarized in table 28-IX. A detailed discussion of all of the extrinsic, echinocytogenic agents identified in the plasma is outside the scope of this presentation. However, the following points should be emphasized relative to the results of this study and the body of knowledge existing relative to echinocyte formation.

The characteristics of the echinocyte formation observed during the Skylab flights are comparable to those of the discocyte-echinocyte transformation induced by elevation of plasma lecithin, lysolecithin, and/or free fatty acids. Most of the echinocytes observed in the Skylab Study were of the stage I type (fig. 28-20) suggesting that the changes in the plasma echinocytogenic factors were moderate. It has been demonstrated by Shohet and Haley (ref. 35) that only a small elevation in the lysolecithin content of the red cell membrane is sufficient to initiate this shape change. The discocyte-echinocyte transformation can occur in seconds when echinocytogenic plasma is added to normal red cells (ref. 37). All of the red cell shape changes, regardless of the cell type or duration of the mission, were almost completely reversed to the preflight levels by the first post-flight sampling period (R+0). Thus the modifications in cell shape, which in some cases had occurred over a 2- to 3-month period, were neutralized within 2 to 3 hours of reentry into the normal gravitational environment of the Earth.

Most changes in red cell shape induced by intrinsic factors and those related to aged red cells are not readily reversible. This observation would support the concept of a change in one of the plasma constituents and its uptake by the cell membrane as being the primary cause of the shape changes.

The magnitude of the red cell shape change was not linearly correlated with any plasma constituent measured in the Skylab studies. However, lecithin, lysolecithin, free fatty acids, and albumin (significant to the clearance of free fatty acids) were not measured in either the in-flight plasma or red cell samples. It has been shown that it is the accumulation of the plasma echinocytogenic agent by the cell membrane which causes the shape change, not merely the addition of the agent to the plasma. This being the case, and because the transformations were all early stages of change, it is possible that extensive chemical analyses of these compounds in the plasma would not provide sufficient information relative to the changes in shape.

The significance of the observed red cell shape transformations during Skylab are not readily apparent. Based upon the exercise performance capacity of the crew as measured in-flight (ch. 21) and based upon their cardiovascular response to the stress of lower body negative pressure (ch. 29) during that time, it seems apparent that these changes in red cell shape do not represent a significant compromise to the ability of the body systems to function normally with respect to adequate blood flow and tissue oxygen demand. However, the impact of alterations in red cell shape with respect to the reduction in circulating red cell mass (ch. 26) might be more significant. Severe deformation of circulating red cells can result in their premature sequestering by the RES, primarily the hepatic and splenic systems (ref. 38). The alteration in red cell shape during space flight might provide a sufficient stimulus to the RES to initiate trapping and eventual removal of these cells from the circulating red cell mass.

Maintenance of normal red cell shape and normal deformability are essential to survival of the cell in vivo. A major function of the RES is to remove from circulation those cells whose structure is abnormal or the membrane too rigid. Since the cells that were examined in this study came from peripheral blood samples, they apparently satisfied the RES criteria for nondestruction. However, the abnormal cells remaining in the circulation may be indicative of a greater degree of shape alteration in other cells which, because of the changes in their structure, had been removed from the circulation. Sufficient data are not available to answer this question with certainty. As stated earlier, all of the echinocytes observed were of the stage I type. Studies on the deformability characteristics of echinocytes produced by extrinsic plasma echinocytogenic factors have shown that there are no significant differences in the deformability of these cells compared to discocytes (ref. 34). It is only when the crenation progresses to a point where change in the membrane results in loss of effective surface area, that the consequences are different, and the cells have a reduction in their deformability. The absence of stage II or stage III echinocytes would seem to indicate that the changes observed were not progressing to further more extensive shape alterations.

The magnitude of the echinocyte formation appears to be related to the duration of the flight with no apparent plateau in the curve depicting the response evident after 82 days. The curve describing the stomatocyte plus knizocyte formation has an optimim value between 20 and 30 days after launch, and by 82 days the percent of these types of cells is comparable to the preflight values. This second type of pattern is consistent with that characteristic of the red cell mass loss during these missions. The loss of circulating red cells was also maximal at 20 to 40 days and decreased after that time. However, the recovery of red cell mass was independent of weightlessness or normal gravity after the initial insult (ch. 26; Johnson and Kimzey, manuscript in preparation). Thus, it is not possible to substantiate a direct relationship between the red cell shape alterations during the Skylab missions to the concomitant loss in red cell mass. However, it is an area that should have further investigation.

The significance of the transformations in red cell shape observed during the Skylab study must be considered relative to the limitation of participation of man in extended space flight missions. The results of this one study are not conclusive with respect to this question. Based on these examinations of red cells in normal, healthy men and based on other Skylab experiment data relative to the functional capacity of the red cells in vitro and the performance capacity of man as an integrated system, the changes observed in this study would not appear to be the limiting factor in determining the stay of man in space. However, the results of this experiment and the documented red cell mass loss during space flight raise serious questions at this time relative to the selection criteria utilized for passengers and crews of future space flights. Serious consideration should be given to testing the effectiveness and reserve capacity of the erythropoietic system in those individuals, and until the questions relative to the specific cause and impact of the red cell shape change on cell survival in vivo can be resolved, individuals with diagnosed hematologic abnormalities should not be considered as prime candidates for missions, especially those of longer duration.

Acknowledgments

The author wishes to acknowledge the technical and scientific assistance of the following individuals without whose contributions this study would not have been possible: V. Anand, W.C. Alexander, M. Brower, L.C. Burns, H. Cantu, E.K. Cobb, B.S. Criswell, J. Dardano, C.L. Fischer, R. Landry, W.C. Levin, H. Owens, S.E. Ritzmann, T. Rogers, C. Tuchman, G.A. Waits, L. Wallace, and D.G. Winkler.

References

1. FISCHER, C.L., C. GILL, J.C. DANIELS, E.K. COBB, C.A. BERRY, and S.E. RITZMANN. Effects of the space flight environment on man’s immune system: I. Serum proteins and immunoglobulins. Aerospace Med., 43:856-859, 1972.

2. KIMZEY, S.L., P.C. JOHNSON, S.E. RITZMANN, C.E. MENGEL, and C.L. FISCHER. Hematology and Immunology Studies. In Biomedical Results of Apollo, pp.197-227. NASA SP-368, 1975.

3. RATNOFF, O.D., and G.B. NAFF. The conversion of C’1S to C’1 esterase by plasmin and trypsin. J. of Exp. Med., 125:337-358, 1967.

4. MULER-EBERHARD, H.J., and E.H. VALLOTA. Formation and inactivation anaphylatoxins. In Second International Symposium on the Biochemistry of the Acute Allergic Reaction, pp. 217-228, K.F. Austen and E.L. Becker, Eds. Blackwell, Oxford, 1971.

5. GOLDSTEIN, I.M., M. BRAI, A.G. OSLER, and G. WEISSMANN. Lysozomal enzyme release from human leukocytes: mediation by the alternate pathway of complement activation. J. Immunol., 111:33-37, 1973.

6. BACH, F.H., and N.K. VOYNOW. One-way stimulation in mixed leukocyte cultures. Science, 153:545-547, 1966.

7. POLLIACK, A., N. LAMPEN, B.D. CLARKSON, and E. DE HARVEN. Identification of human B and T Iymphocytes by scanning electron miscroscopy. J. Exper. Med., 138:607-624, 1973.

8. LIN, P.S., and D.F.H. WALLACH. Surface modification of T-lymphocytes observed during rosetting. Science, 184:1300-1301, 1974.

9. KAY, M.M., B. BELOURADSKY, K. YEE, J. VOGEL, D. BUTCHER, J. WYBRAN, and H.H. FUDENBERG. Cellular interactions: Scanning electron miscroscopy of human thymus-derived rosette-forming lymphocytes. Clin. Immunol.Immunopathol., 2:301-309, 1974.

10. FAUCI, A. S., and D.C. DALE. The effect of in vivo hydrocortison on subpopulations of human lymphocytes. The J. of Clin. Invest., 53:240-246,1974.

11. MARKMAN, M.H. Properties of adenylate cyclase of lymphoid cells. Proc. Nat. Acad. Sci., 68:885-889, 1971.

12. SMITH, J.W., L. STEINER, and C.W. PARKER. Human lymphocyte metabolism. Effects of cyclic and noncyclic nucleotides on stimulation by phytohemagglutinin. J. Clin. Invest., 50:442-448, 1971.

13. COOPER, H.L., and H. GINSBURG. Lymphocyte activation II. In Progress in Immunology, pp. 1147-1152, B. Amos, Ed. Academic Press, New York, 1971.

14. DANON, D., and Y. MARIKOVSKY. Determination of density distribution of red cell population. J. Lab. Clin. Med., 64:668-674, 1964.

15. KIMZEY, S.L., S.E. RITZMANN, C.E. MENGEL, and C.L. FISCHER. Skylab experiment results: Hematology studies. Acta Astronautics, pp. 141-154, 1975.

16. LARKIN, E.C., and S.L. KIMZEY. The response of erythrocyte organic phosphate levels and active potassium flux to hypobaric hyperoxia. J.Lab. Clin. Med., 79: 541-549, 1972.

17. HERZ, F., and E. KAPLAN. A microtechnic for the separation of erythrocytes in accordance with their density. Amer. J. Clin. Path., 43:181-183, 1965.

18. BESSIS, M., R.I. WEED, and P.F. LEBLOND. Red Cell Shape: Physiol. Pathol., Ultrastructure. Springer Verlag, New York, 1973.

19. BRECHER, G., and M. BESSIS. Present status of spiculed red cells and their relationship to the discocyte-echinocyte transformation: a critical review. Blood, 40:333-344, 1972.

20. BULL, B.S., and I.N. KUHN. The production of schistocytes by fibrin strands (a scanning electron microscope study). Blood, 35:104-111, 1970.

21. COOPER, R.A. Anemia with spur cells: a red cell defect acquired in serum and modified in the circulation. J. Clin. Invest., 48:1820-1831,1969.

22. COOPER, R.A., and J.H. JANDL. Bile salts and cholesterol in the pathogenesis of target cells in obstructive jaundice. J. Clin. Invest.,47:809-822, 1968.

23. KAYDEN, H.J., and M. BESSIS. Morphology of normal erythrocyte and acanthocyte using nomarski optics and the scanning electron microscope. Blood, 35:427-436, 1970.

24. ADAMS, K.H. Mechanical equilibrium of biological membranes. Biophys. J., 2:123-130, 1972.

25. ADAMS, K.H. A theory for the shape of the red blood cell. Biophys. J., 13:1049-1053, 1973.

26. BULL, B.S. Red cell bioconcavity and deformability, a macro-model based on flow chamber observations. In Red Cell Shape, pp. 115-124, M. Bessis, R.I. Weed, and P.F. Leblond, Eds. Springer Verlag, New York, 1973.

27. BULL, B.S., and J.D. BAILSFORD. The biconcavity of the red cell: an analysis of several hypotheses. Blood, 41:833-844, 1973.

28. EVANS, E.A., and P.F. LEBLOND. Image holograms of single red blood cell discocyte spheroechinocyte transformation. In Red Cell Shape, pp. 131-140, M. Bessis, R.I. Weed, and P.F. Leblond, Eds. Springer Verlag, New York, 1973.

29. KIMZEY, S.L. Special hematologic effects (M115). In Skylab Medical Experiments Altitude Tests, pp. 6-21 through 6-32. NASA TM X-58115, 1973.

30. BESSIS, M., and L.S. LESSIN. The discocyte-echinocyte equilibrium of the normal and pathologic red cell. Blood, 36:399-403,1970.

31. DEUTICKE, B. Transformation and restoration of biconcave shape of human erythrocytes induces by amphiphilic agents and changes of ionic environment. Biochim. Biophys. Acta, 163:494-500,1968.

32. FEO, C. The role of lysolecithin formed in plasma on the discocyteechinocyte transformation, a commentary. In Red Cell Shape, pp. 37-40, M. Bessis, R.I. Weed, and P.F. Leblond, Eds. Springer Verlag, New York, 1973.

33. LA CELLE, P.L., F.H. KIRKPATRICK, M.P. UDKOW, and B. ARKIN. Membrane fragmentation and Ca-membrane interaction: potential mechanisms of shape change in senescent red cell. In Red Cell Shape, pp. 69-78, M. Bessis, R.I. Weed, and P.F. Leblond, Eds. Springer Verlag, New York, 1973.

34. LEBLOND, P. The discocyte-echinocyte transformation of the human red cell: deformability characteristics. In Red Cell Shape, pp. 95-104, M. Bessis, R.I. Weed, and P.F. Leblond, Eds. Springer Verlag, New York,1973.

35. SHOHET, S.B., and J.E. HALEY. Red Cell membrane shape and stability: relation to cell lipid renewal pathways and cell ATP. In Red Cell Shape, pp. 41-50, M. Bessis, R.I. Weed, and P.F. Leblond, Eds. Springer Verlag, New York, 1973.

36. WEED, R.I., and B. CHAILLEY. Calcium—pH interactions in the production of shape change in erythrocytes. In Red Cell Shape, pp. 56-58, M. Bessis, R.I. Weed, and P.F. Leblond, Eds. Springer Verlag, New York, 1973.

37. BESSIS, M. Living Blood Cells and Their Ultrastructure, pp. 146-155. Translated by R.I. Weed). Springer Verlag, New York, 1973.

38. RIFKIND, R.A. Destruction of injured red cells in vivo. Amer. J. Med., 41:711-723, 1966.

 

	JSC Home Page   NASA Home Page
	What you need to know about NASA JSC Web Policy
	Curators: Afzal Ahmed  and Julie Oliveaux
	Responsible NASA Official: Judith L. Robinson, Ph.D.
	Several NASA centers participate in the Life Sciences Data Archive project: Judith L. Robinson, Ph.D., LSDA Project Manager Paul X. Callahan, Ph.D., Data Archive Project Manager at NASA Ames Research Center (ARC) Judith L. Robinson, Ph.D., Data Archive Project Manager at NASA Johnson Space Center (JSC) Bridgit O'Hara Higginbotham, Data Archive Project Manager at Kennedy Space Center (KSC)
	Last Modified