TheClinicalChemistryandImmunologyofLong … · 2017-04-13 · CLIN.CHEM.39/1,22-36(1993)...

CLIN.CHEM.39/1, 22-36 (1993)

22 CLINICALCHEMISTRY,Vol.39, No.1, 1993

The Clinical Chemistry and Immunology of Long-Duration Space MissionsAlan II. B. Wu,”3 Gerald R. Taylor,2 Gary A. Graham,3 and Bruce A. McKinley4’6

Clinical laboratory diagnostic capabilities are needed toguide health and medical care of astronauts during long-duration space missions. Clinical laboratory diagnostics,as defined for medical care on Earth, offers a model forspace capabilities. Interpretation of laboratory results forhealth and medical care of humans in space requiresknowledge of specific physiological adaptations that oc-cur, primarily because of the absence of gravity, and howthese adaptations affect reference values. Limited datafrom American and Russian missions have indicatedshifts of intra- and extracellular fluids and electrolytes,changes in hormone concentrations related to fluid shiftsand stresses of the missions, reductions in bone andmuscle mass, and a blunting of the cellular immuneresponse.These changes could increasesusceptibilitytospace-related illness or injury during a mission and afterreturn to Earth. We review physiological adaptations andthe risk of medical problems that occur during spacemissions. We describe the need for laboratory diagnosticsas a part of health and medical care in space, and howthis capability might be delivered.

AddItIonal keyphrases: space medicine effects of micro-gravity acute myocardial infarction nephrolithiasis . elec-trolytes . osmolality bone bacteria immune status

For humans to migrate from Earth to other parts ofthe solar system, essential parts of the Earth environ-ment must be provided and some adaptation of the bodyto the space environment must occur. Factors that affecthuman physiology include the physical and psychologi-cal stresses associated with the mission, the duration ofthe mission, and the space vehicle or habitat. Earth-normal gravity and the protection from space radiationprovided by the Earth’s atmosphere are among thefactors that are the most difficult to duplicate. Inappro-pnate attention to these factors could produce acute orchronic health problems. Radiation hazards beyond lowEarth orbit are known to include solar particle events

‘Department of Pathology and Laboratory Medicine, Universityof Texas Medical School, Houston, TX 77030.

2Mail code SD5, Johnson Space Center, Houston, TX 77058.

3Eastman KOdak Co., Rochester, NY 14650.KRUG Life Sciences, Inc., Houston, TX 77058.Current address (address for correspondence): Hartford Hospi-

tal, Clinical Chemistry Laboratory, Hartford, CT 06115.6Cuirent address: Department of Anesthesiology, University of

Texas Medical School, Houston, TX 77030.Presented in part at the Clinical Chemistry in Space Sympo-

sium, Texas Section of AACC, Houston, TX, March 1990.Received July 2, 1991; accepted October 26, 1992.

and galactic cosmic radiation, but are not well charac-terized by type, exposure risk, or physiological effects(1). Experience of Earth-orbital missions has shownthat absence of gravity causes important physiologicalchanges. The time course and extent of these changesduring long-duration space missions are not known-primarily because of limited experience, both in num-bers of individuals and duration of missions (2). Also,since Skylab in the early 1970s, the capabilities forinvestigating physiological adaptations on long-dura-tion missions have been limited to the Russian spaceprogram. Although investigations of specific cardiovas-cular and neurological adaptations and countermea-sures have been an important part of recent space-shuttle orbiter missions, standard clinical laboratoryanalyses performed in space have not been available. Inrecent years, however, technology has become availablethat can be used to provide clinical laboratory diagnosisin space.

Health and MedIcal Care durIng Long-Duration

MIssIons

Long-duration missions, i.e., >10 days in space, arean important part of NASA’s future plans for humanexploration of space. American long-duration missionsare now planned to extend to 16 days with NASA’s spaceshuttle orbiter (Shuttle), to 90 days with Space StationFreedom (5SF), and possibly to 1000 days for explora-tion missions of the moon and Mars.7 The Russian SpaceStation Mir has provided long-duration missions of 6months to 1 year for one to three crew members.

Physiological adaptations to space are studied be-cause of concern for the health of astronauts and todetermine the need for and effectiveness of appropriatecountermeasures. For short-duration missions, thehealth and safety of astronauts are assured somewhatby intensive crew selection and training procedures,extensive inspection of space systems, and the capabil-ity for immediate return to Earth. However, investiga-tion of the long-term effects of space exposure is needed.Health care needed during long-duration missions is notyet defined. The extent of instances of and lack of knownmeasures to counter muscle atrophy, loss of bone mass(possibly irreversible), cardiovascular deconditioning,

7Nonstandard abbreviations: NASA, National Aeronautics andSpace Administration; SSF, Space Station Freedom; HMF, HealthMaintenance Facility; ICU, intensive-care unit; GFR, glomerularfiltration rate; ASTP, Apollo-Soyuz Test Project; CK, creatinekinase; CMI, cell-mediated immune; DTH, delayed-type hypersen-sitivity; IL, interleukin; and INF-g, interferon gamma.

CLINICALCHEMISTRY, Vol. 39, No. 1, 1993 23

cardiac arrhythmias, radiation damage, and other ef-fects are important health-care issues.

Beginning with 13-day missions in 1992-3, Shuttlemission duration is planned to be increased to 16 daysfrom the current maximum of 11 days, as a goal of theExtended Duration Orbiter program (3). The purposesof this program are to provide more on-orbit laboratorytime for life science experiments in micro-gravity andspace and to support construction of SSF. Plannedinvestigations of human physiological adaptation in-clude cardiovascular regulation of fluids and electro-lytes, exercise and muscle performance, neurosensoryand sensorimotor control, muscle atrophy, renal stoneformation, nutrition, and environmental health (4).

The Shuttle Orbiter Medical System is provided formedical care of the crew during current missions. This

medical system includes two kits of medical suppliesand instruments stowed aboard each Shuttle. For ex-tended-duration operations, a third kit would be added.During a mission, either of two crew members trainedas crew medical officers could provide care. An Earth-based NASA physician would be the crew surgeon, andwould have overall responsibility for crew health andmedical care before, during, and after the mission.Other Shuttle medical operations include premissioncrew health stabilization (partial crew isolation 7 daysbefore the launch), pre- and postmission physical exam-inations (including collection of blood and urine sam-ples), instruction and training of the mission crew anddesignated crew medical officers in medical procedures,and on-orbit detection and management of toxic sub-stances (5, 6).

The SSF program was revised during 1990-1 andcurrently plans man-tended capability by the end of1996 and permanent manned capability by the end of1999. Man-tended capability would permit 12- to 30-daymissions for crews of as many as five members. AShuttle would provide transportation to and from SSFand would remain docked at the station throughout themission. Missions as long as 90 days for four-membercrews are currently envisioned in permanent mannedcapability, with crew rotations at regular intervals.Detailed investigations of human physiological adapta-tion to space are planned aboard SSF (7).

A Crew Health Care System for SSF has been inplanning and design since 1983. The Crew Health CareSystem comprises an Environmental Health System, anExercise Countermeasures Facility, and a Health Main-tenance Facility (HMF) (8). At man-tended capability,the Shuttle Orbiter Medical System will be used forminor medical problems and the HMF will providelimited equipment and supplies for advanced life sup-port, including cardiac defibrillation and monitoring,ventilatory support, and fluid-replacement therapy suf-ficient for medical transport to Earth via a Shuttle. Atpermanent manned capability, a Shuttle would notalways be docked at SSF and would not be readilyavailable for return of the crew to Earth. Plannedadditions to the HMF are therefore to include capabili-ties for laboratory diagnosis, physiological monitoring,

intravenous fluid formulation, surgery, and dental care.These additions would permit resuscitation, stabiliza-tion, and intensive care of a seriously ill or injured crewmember at SSF for as long as 3 days. Hyperbarictreatment capability is planned as part of extravehicu-lar activity airlock systems. Medical transport capabil-ity from SSF to a definitive medical care facility onEarth within 24 h of a decision to transport is plannedvia an Assured Crew Return Vehicle (9).

Human exploration of the moon and Mars is in earlyplanning (10, 11). The current focus is a First LunarOutpost mission of 45 days. Planned lunar missions of14 days to 2 years and Mars missions of 500-1000 dayshave been described. The primary activity of explorerswould be “fieldwork” to chart and examine the planet’ssurface, obtain mineralogical and water samples, andset up and maintain astronomy equipment and addi-tional outpost facilities. The work would involve exten-sive use of protective pressurized suits and portablelife-support systems. The health and performance ofeach exploration crew member are basic to its planningand will be essential for mission success.

Return to Earth would require several days from alunar outpost and several weeks to months from a Marsmission. The delivery of medical care on site wouldtherefore be a very important operational capability forexploration missions, because the need to return anindividual to Earth for medical care would require thatthe entire crew return, the mission be aborted, and theoutpost be (temporarily) abandoned. Also, a prognosticcapability that at present does not exist would be neededto plan a successful long-term transport. To plan asurvivable medical transport of a crew member from alunar outpost, we must be able to project over severaldays the clinical course and condition of a crew member.Physiological adaptation to self-contained, micro- andpartial-gravity environments, as well as radiation haz-ards of exploration missions, could introduce health andpotential medical care concerns that would requireperiodic or continuous monitoring. Illness or injury,possibly complicated by adaptation stresses, wouldtherefore require prepared, informed response. Conceptsof health and medical-care systems for space explorationhave been described, based primarily on the technicalevolution of current concepts (12).

Laboratory DIagnostic RequIrements for MedIcal CareIn Space

Clinical medical care during long-duration spacemis-sions is emerging as an important part of programplanning (13-16). Laboratory diagnosis will also benecessary for medical care in space. Because certain

health problems may be exaggerated by conditions ofthe space environment, health and medical care may beclosely linked. The same laboratory analytical capabil-ity will probably have an important role in monitoringphysiological adaptation and the necessity and effec-tiveness of countermeasures.

Laboratory analysis in space medicine will probablyrely on proven, standard technology and analytes, be-

24 CLINICAL CHEMISTRY, Vol.39, No. 1, 1993

cause the technology will be available for adaptation tothe space application, the knowledge of analyte physi-ology in Earth-based practice is extensive, and thebudgets for basic research and development of newsensors will be limited. Definition of laboratory diagnos-tic capability for space medical operations and researchis likely to evolve from a known Earth-based exampleby analogy, and without requirements established by alarge patient population in space. Critical care haswell-defined laboratory diagnostic requirements thatare a major part of clinical laboratory capabilities (17).Use of the laboratory for critical care on Earth may haveimportant similarities to its use in medical care atremote self-sufficient outposts in space.

Need for Critical-Care Capability

Serious injury or illness of a crew member during along-duration exploration mission would have conse-quences for that individual and for the mission. Mission-related consequences might include mission disruptionbecause of the involvement of other crew members withcaring for the victim (short- and long-term), loss of(some) mission objectives because of full or temporaryincapacitation of part of the crew, and (or) missiontermination because of an inability to resolve the situ-ation. Consequences for the ill or injured individual(s)would include recovery with appropriate care alter sev-eral days of incapacitation, partial recovery after sev-eral weeks of incapacitation with possible permanentdisability, or death within hours to several weeks be-cause of the illness, injury, or its complications. Exceptfor a massive injury resulting in immediate death, all ofthese consequences could be minimized with medicalexpertise, communication, equipment, and supplies im-mediately available as part of the mission operationalcapability and crew. The crew surgeon, responsible formedical care of the crew, could provide necessary med-ical expertise. Any serious medical problem would re-quire rapid development of a clinical diagnosis and atreatment plan. A less serious injury or illness wouldrequire similar monitoring and planning to preventprogression to a life-threatening illness. Diagnostic in-formation, especially if obtained, analyzed, and commu-nicated efficiently, would be the basis of decisions fortreatment and clinical management (18).

Laboratory diagnostic capability as a part of themedical-care system aboard the space vehicle and at theplanet surface habitat would be a major part of diagnos-tic capability for the crew surgeon. In combination withmedical informatics (also as part of the medical systemaboard the space vehicle and the planet surface habitat)and consultation with Earth-based specialists, accurate,clinically current decisions could be made. Laboratorydiagnosis, established remotely from Earth or locally atbedside, depending on expertise of the crew surgeon,would be based on comparison of recent clinical labora-tory variables over time, adapted normal physiologicalranges of specific analytes, and other clinical informa-tion. Laboratory analysis may be the most direct, easilyaccomplished, and informative of the options available

for establishing a diagnosis and monitoring the clinicalcourse.

The importance of clinical laboratory information inmanagement of critically ill patients on Earth may offerinsight to its potential importance at a remote outpostin space. Recent estimates of clinical laboratory use intertiary-care hospitals show that 20-25% of the labora-tory workload is stat orders, mostly from critical care(19), and that 62% of laboratory costs is from criticalcare (20). Chemistry and blood gas data were used in42% of clinical decisions in surgical intensive-care unit(ICU) rounds (21). An average of 23 analyses per pa-tient-day, including blood gas and hematology but ex-cluding microbiology, were ordered in another surgicalICU (17). A study of use of laboratory results requestedin an ICU during night or weekend hours found that34% led directly to changes in patient therapy, that 71%were “clinically helpful,” and that a relatively highdegree of consultant staff involvement may have con-tributed to efficient use of laboratory services and re-sults (22). Clearly, critical-care requirements signifi-cantly affect laboratory capability in Earth-basedhospitals; when illness or injury is serious, laboratoryresults are also critical.

Considerationsfor Designof ClinicalLaboratoryDiagnosticEquipment and a Medical-Care System for SpaceExploration Missions

Planning medical-care capability for a space-explora-tion mission requires examination of current (Earth)practice, the standard of care to be provided, the poten-tial patient population and environment, and anyunique hazards of the space environment to whichexplorers would be exposed. Far in advance of anyspecific mission and with limited experience in theenvironment to be explored, the most applicable experi-ence is current, terrestrial medical care. The mostsignificant medical risks to healthy individuals who are

expected to make up exploration mission crews aretrauma and infectious disease (18). Responding to life-threatening instances of trauma and infectious diseaseusually requires critical care in versatile, dedicatedfacilities that are based on individual patient bed unitsand patient care assignments; frequently, emergencysurgical care in the same or similar facilities is required.

Components of emergency surgical and critical-carecapability in addition to physician and nursing careinclude laboratory and imaging diagnostics, centralsupply, and pharmacy, respiratory therapy, physicaltherapy, and other services. In contrast with currentEarth-based critical care, extreme limitations of volumeand weight of equipment and supplies and of the num-ber of personnel available to deliver care are likely.Patient capacity of a medical-care system in space is animportant design factor, particularly when the volumeand mass of equipment and supplies are severely re-stricted. One patient bed unit is planned for a crew offour to eight at a planet surface outpost, with duplica-tion if necessary (12). Efficiency of the caregiver(s),including ready access to equipment and supplies, will

Table 1. PriorIties for Laboratory Diagnostic CapabilItyPriority Assay Priority

Serum chemistryUnc acid 2y-Glutamyltransferase 2Creatine kinase 2

11

1

111

11

Priority

1

112222

1

1111

1113

CLINICALCHEMISTRY,Vol.39, No.1, 1993 25

be necessary to provide such care. The crew surgeon willneed to have a full working knowledge of all resourcesavailable and will have to use them optimally to deliverappropriate medical care. Clinical laboratory and imag-ing diagnostic capabilities available at or near thepatient would be important resources.

Laboratory diagnostic capability is planned to beavailable at bedside. The rationale for this is identical tothat for bedside clinical chemistry in Earth-based ICUoperations: setting priorities for analysis and care by thecaregiver, minimizing sample volume and eliminatingtransport to a laboratory remote from the patient, mm-iinizing turnaround time from the ordering of tests tothe decision to act on results, and interpreting resultswithin the bedside clinical context.

Several factors influence the design of laboratorydiagnostic equipment for space- or Earth-based applica-tions. Considering bedside critical care as a “worst case”

for design, all phases of critical care can involve crisisresponse, and the need for clinical chemistry and bloodgas data can be greatest at these times. Automation ofclinical chemistry and other laboratory diagnostic func-tions could offer significant benefit to a crew surgeonfaced with multiple tasks to be accomplished simulta-neously. Test menu and throughput are important butconflicting design factors, because an extensive menucould require a rapid rate of sequential analysis. Expe-rience with various analyzers that provide only a lim-ited number of analyses, e.g., blood gas and electrolyteanalyzers in ICUs and recovery rooms of 10-30 beds,indicates that a throughput rate of <100 analyses perhour is adequate (23). However, the availability of testsfor 20-40 analytes could require a substantially higherthroughput rate. The time required for calibration,performance check, and preventive maintenance proce-dures and the volume and mass of the supplies wouldneed to be minimized.

Development of instrumentation and operating proce-

dures for a specific application, e.g., clinical chemistry,requires technical development, trial, and error. Capa-bility can be specified on the basis of current technolo-gies and procedures, as initial efforts for the SSF HMFdemonstrated (24,25).

Developments for Space Station Freedom Health

Maintenance Facility

Before a recent redesign, the medical-care system foruse at SSF was designed to provide care for a crew of asmany as eight for as long as 45 days (26). Laboratoryanalytical requirements for the remote system weredetermined in cooperation with a consultant group ofphysicians and biomedical engineers representing sev-eral medical specialties, e.g., anesthesiology, criticalcare, general surgery, internal medicine, medical in-

formatics, and pathology. Prioritization of needs forspecific laboratory analyses routinely available andcommonly used in standard medical practice was estab-lished according to the following criteria:

#{149}Priority 1. Essential for diagnosis, determinationand guidance of therapy, and monitoring of clinicalcourse.

#{149}Priority 2. Desirable for diagnosis, therapeutic deci-sion-making, and confirmation.

#{149}Priority 3. Infrequently used for key clinical deci-sion-making.

The priority assigned to each analyte is listed in Table1. Not surprisingly, to address the potentially worst(medical) case for the crew of a space station mission,clinicians who rely on laboratory results for clinicaldecision-making recommended that the capability of acomplete clinical laboratory be available at a spacestation outpost. This recommendation, modified forchanges in clinical practice and new analyses, wouldprobably also apply to a planetary outpost or any ex-tended-duration exploration mission for which standardmedical care would be provided.

AssaySerum chemistryElectrolytesBloodgasesCalciumPhosphorusGlucose

Total and direct bilirubinCreatinineBlood urea nitrogenTotal proteinAlbuminAlanineaminotransferaseAspartate aminotransferaseAmylaseAlkalinephosphataseMagnesiumLactateCholesterol

IronTriglycerides

Hematology/coagulatIon

Complete blood countPlateletsProthrombintimePartial thromboplastin time

1 Cerebrospina! fluid1 Glucose1 Protein2 Leukocytes2 Culture2 Relative density

Assay

Urine analysispHElectrolytesCreatinine

3 Urea nitrogen3 Relativedensity

Calcium

1 Phosphorus1 Total protein1 Amylase

Erythrocyte and leukocyte1 counts

Microbiology culturesBlood, urineNose, throatWound,sputum,stoolAntibioticsensitivity

B.

HEIGHTINCREASE

1.3 cmINCREASED SUSCEP11BILIrYTO INFECTION

LOSSweiit-being

WEIGHT3.4% loss

2l3waterll3body& t loss

1-g


Clinical chemistry was noted to include -75% of thelaboratory analytes recommended and was pursued asan initial development project for the SSF HMF. Afterextensive investigation of developing technologies andcommercially available systems, an experimental chem-istry analyzer system was designed and built in 1988-9by Eastman Kodak Co. (Rochester, NY) to address spaceclinical chemistry requirements (24). The analyzer wasdesigned to provide 32 standard clinical chemical anal-yses of discrete blood, urine, or cerebrospinal fluid sam-ples, i.e., all priority 1 and many priority 2 chemicalanalytes. System and operational issues were also ad-dressed, including volume and mass of equipment andsupplies, electrical power requirement (and other ser-vice utilities), crew time for use of equipment, reliableautomation of procedures, commercial availability ofdevices and (or) technology, modular design (multiplefunctions, replaceable parts), and reliability of supply,service, and long-term upgrades. The performance ofthis experimental system is described further by Tonne-sen, Wu, McKinley, et al. (unpublished) and in relatedpublications (24,25).

Currently, clinical chemistry and blood gas analysesare planned to be provided as parts of the HMF tosupport permanent manned capability in late 1999.Clinical microbiology capability is planned to be avail-able with the Environmental Health System. This sys-tem would have the primary purpose of detection oftoxic substances or microbiological contamination of theinternal habitable environment of the space station, butwould include instrumentation with clinical application(26). Microbiology capability is planned to be based on

A.

microscopy and automated culture analysis (AutoMicro-bic System; Vitek Systems Inc., St. Louis, MO). Thissubsystem might not be located near the HMF, so thatsample transport from a patient to this facility would benecessary.

Physiological Adaptations to Space and Effects onClInIcal ChemIcal Variables

Many physiological changes occur before, during, andafter humans are exposed to space (see Figure 1). Thesechanges are initiated by the fluid shifts that occurbecause of the absence of Earth-normal gravity and thestress associated with space flight (27). Fluid and elec-trolyte movement between vascular, extracellular, andintracellular fluid compartments over a period of hourscontributes to cardiovascular deconditioning, an alteredstate of the cardiovascular system that is characterizedby reduced cardiac output and lowered reflex sensitivi-ties (28). Reduced cardiac output may affect the bio-availability of drugs. Disturbances in electrolyte con-centrations may increase the risk of nephrolithiasis.Endocrine changes related to prelaunch stress maycontinue during adaptation to microgravity. Prolongedlack of gravity and musculoskeletal loading causesskeletal muscle and bone atrophy, which, together withspace radiation, may present the most serious threats tohealth of crew members during long-duration missions.Such ongoing changes could affect results and interpre-tations of routine clinical chemical analyses.

ElectrolyteTestingin Relation to Fluid Shifts

The redistribution of fluids as a result of weightless-

ness has significant effects on water and electrolyte

C.

ENDOCIINE RESPONSE HORMONALTOMISSIONSTRESS DERANGEMENTS

IWvIUNE

‘ ARRHYTHMLaS

_____ ELECTROLYTE IMBALANCE,KiDNEYSTONES

FRACTURES, OSTEOPOROSISWEAKNESS, FATIGUE

O-g Medical ConsequenceFig. 1. Summaryofphysiologicalchangesdue to microgravity exposure: (A) 1-g environment; (B) O-g environment; (C) medical consequencesof physiological adaptations

/20 40 60

a

40

a

uo

Preflight1-6 14-18

Postflight

CLINICALCHEMISTRY, Vol.39, No. 1, 1993 27

balance (29). An estimated 0.5-3 L of blood is rapidlyredistributed between the lower extremities and thecentral blood volume. Preliminary results of the recentSpacelab Life Sciences 1 mission indicate that fluidshifting occurs before and soon after launch (30). Cen-tral osmo- and neuroreceptors perceive this shift as anincrease in the total blood volume. Under Earth-normalgravity conditions, volume expansion leads to diuresis,a decrease in the thirst sensation, and an increase in theglomerular filtration rate (GFR). During Skylab, GFRestimated by creatinine clearance was shown to beincreased during space ifight (31). However, becauseserum creatiine concentration was also increased (32),possibly as the result of skeletal muscle turnover, theindication of increased GFR may have been inaccurate.Nevertheless, inulin studies during Spacelab Life Sci-ences 1 also showed that GFR increased under Earth-normal gravity conditions (28). In contrast, renalplasma flow, as measured by plasma p-aminohippurate(33), decreased (30), which may increase the risk fornephrolithiasis (see below).

The shift of blood volume from the extremities, espe-cially the legs, is apparently sensed as an increase inblood volume. Adjustment of blood volume results indiuresis and depletion of electrolytes and total bodywater. Results from Skylab showed serum sodium activ-ity inf light to be less than that during preffight, whereaspotassium activity was slightly increased (Figure 2).These adaptations are consistent with the observeddecrease in the release of antidiuretic hormone (Figure2), renin, and aldosterone, and an increase in atrialnatriuretic factor. However, during Spacelab Life Sci-ences 1, atrial natriuretic factor decreased by 20%during the first 6-8 h in orbit, indicating that this factoris probably not involved with the early diuresis ob-served (28).

Because vascular volume redistribution is due to ashift of fluid toward the head and not to an actualincrease in blood volume, astronauts are usually dehy-drated during the first few days of a mission, and aconscious effort is made to maintain adequate waterintake. Indeed, most Skylab astronauts lost weight,mostly attributable to a negative water balance, asdetermined by intake and excretion measurements (32).

The cardiovascular adaptation of fluid shifts in micro-gravity includes a decrease in heart size, stroke volume,and left ventricular end-diastolic volume. The resettingof these sympathetic regulatory mechanisms producesan orthostatic intolerance upon return to Earth, includ-ing tachycardia and a syncopal decrease of blood pres-sure. These effects can last for 2-3 days after returningto Earth. An effective countermeasure to this intoler-ance is fluid loading with saline just before re-exposureto gravity. However, excessive fluid replacement andloading early in a long-duration mission may retardnormal cardiovascular adaptation and may be counter-productive (29).

For long-duration space missions, analysis of serumand urine for electrolytes should be a vital part ofroutine health monitoring, in addition to monitoring the

status of these analytes during acute injury or illness.The capability for measurement of serum sodium, po-tassium, and chloride activities-as components of os-motic fluid balance-could be particularly important.The capability for measurement of magnesium concen-tration could provide important information about nu-tritional status.

Total CO2 measurements would also be a reliableindicator of buffering capacity and ventilatory status. Incombination with arterial and venous whole-blood pHanalysis, measurements of airway end-tidal CO2 gasconcentration measurement and lung mechanical func-tion would provide an accurate assessment of ventila-tion. The capability for complete blood gas analysis, toinclude P02, Pco2, pH, and hemoglobin, in combinationwith inspired and expired gas analysis and lung me-chanical functions, would permit assessment of oxygen-ation, ventilation, and work capacity.

Osmotic Measurements for Health Monitoring

Serum osmolality has important applications in mon-itoring treatment of cerebral edema and chemical intox-ication. Confirming and monitoring multiple organ fail-ure have been reported, with the correlation of thedifference between measured and calculated osmolalityattributed to leakage of amino acids and undetermined,intracellular solutes, the result of cellular or tissuedamage (34). In the event of a serious head injury to acrew member that resulted in cerebral edema and anincrease in intracranial pressure, treatment could relyon pharmacological adjustment of plasma and cerebro-spinal fluid osmolality, guided by osmolality measure-

0

EE

ILj

0(1)

a io1601

_

2 40 80010

1-28 29-59 60-85InflightDAYS

Fig. 2. Average of plasma sodium, cortisol, and 24-h urine anti-diuretic hormone results from pre-, in-, and postfllght collections bySkylab 2, 3, and 4 astronauts (30)mtlight sampleswere stored frozen andassayedafter return to Earth

28 CLINICAL CHEMISTRY, Vol. 39, No. 1, 1993

ments and, if possible, monitoring of intracranial pres-sure. Hyperventilation or surgical intervention mightbe effective, but would be highly dependent on theexpertise of the crew surgeon. Elevation of the head inpartial- or microgravity would not be effective, and theaccelerations associated with transport might not besurvivable. Osmolality analysis can provide a rapid,convenient indication of electrolyte and metabolite nor-malcy and, compared with an estimate based on analyt-ical data for sodium, glucose, and urea, can be used toindicate the presence of another, possibly toxic osmoticsolute(s) (35). Because physiological adaptation to spacedoes not involve abnormal constituents of plasma, os-molality of plasma and interstitial fluid would be ex-pected to remain within the normal range. However,acute effects of space adaptation, e.g., severe spacemotion sickness with emesis, could cause changes inosmolality, which could be easily monitored. Confirmingdehydration, tailoring hydration fluids in preparationfor normal gravity or intense extravehicular activity,monitoring renal function, and detecting radiation dam-age through low-molecular-mass intracellular contentsare other potential uses of osmolality measurementsduring long-duration, space exploration missions thatcould be important to health care and the prevention ofthe need for medical care.

Plasma colloid osmotic pressure is known to decrease

from 25 to -21.5 mniHg with a change in posture fromupright to supine (36). The change in posture in Earthgravity causes a change in fluid column height and theresorption of interstitial, hypo-osmotic fluid from the legsinto the vascular space, a change that is similar to thatencountered upon exposure to the microgravity of space.Critically ill patients frequently have decreased plasmacolloid osmotic pressure. Malnutrition, trauma, hemor-rhage, inflammation, infusion of large volumes of crystal-loid fluid, and malignancy can produce hypo-osmoticstates because of the catabolism or dilution of albumin andother osmotically active proteins. Although extremechanges of colloid osmotic pressure may not occur withphysiological adaptation to space, modest changes similarto those due to postural changes may occur and be exag-gerated by stress, forced intake of water or crystalloidsolutions, and the disruption of food intake. Such changes

could predispose astronauts to medical problems. Mea-surement of colloid osmotic pressure, as part of healthmonitoring for long-duration space missions, could provideimportant information describing the general health ofindividual crew members.

Electrolyte Testing in Relation to Bone Loss

Bone demineralization and the subsequent loss ofbone mass due to absence of Earth gravity may presenta serious health-care problem for long-duration, spaceexploration missions (37). Bone is a dynamic tissueundergoing a continuous formation and resorption pro-cess referred to as remodeling. This remodeling is acomplex process involving calcium and phosphorus me-tabolism, local influences, and humoral endocrine regu-lators (38). Indeed, some disturbance of one or more of

these factors may account for the bone loss observed inspace. Whatever the mechanism, the result is that boneresorption exceeds bone formation in the absence ofgravity, much as it does in the aging of humans onEarth after the fourth or fifth decade of life. Spaceosteopenia is similar to osteoporosis, in that the remain-ing bone has a normal ratio of mineral to organic tissue.Although bone loss has been observed on all long-term(s3 months) American space missions, no astronaut hashad impaired bone function as a result (39, 40). How-ever, the unknown consequences of longer exposure toweightlessness and the consequences of returning toEarth gravity after this exposure make bone loss apotentially serious problem that must be addressed.

In addition to the loss of bone mass, humans in spaceexperience a negative calcium and phosphorus balance,resulting primarily from excessive urinary excretion ofthese minerals (39). It is not clear whether this distur-bance in calcium and phosphorus metabolism is a pri-mary or a secondary effect of bone demineralization,which increases the prerenal load of these minerals.Endocrine studies during the Apollo and Skylab mis-sions showed no changes in parathyroid hormone, calci-tonin, or 25-hydroxycholecalciferol that would accountfor bone demineralization during space ifight (40-42).Changes in serum concentrations of thyroxine, insulin,and urinary glucocorticoids also do not conclusivelyaccount for the loss of bone.

One possible cause of space osteopenia may be thelack of mechanical loading stress on bones. Stress is oneof several so-called “local influences.” Stress produceselectrical fields in bone that stimulate osteoblastic ac-tivity (39). The lack of stress would therefore be ex-pected to reduce the rate of bone formation relative toresorption. Prostaglandin E2 has been implicated inmediating the effect of stress on bone formation (43).Another observation that supports a possible role forstress is that the bone loss is greatest from the weight-bearing bones (41, 44, 45). However, no observation todate eliminates the possibility of a primary increase inbone resorption as a cause of bone loss in space.

Recent studies in which prolonged bed rest was usedas a model for weightlessness demonstrated significantbiological variation in the individuals studied (46). Therate of total body calcium loss was greater in this studythan that observed in space ifight. The percentagechange in bone density varied greatly among the differ-ent bones evaluated, with the calcaneous losing 0.6%per week and the distal radius losing 0.03% per week.The rate of mineral recovery after reambulation alsovaried by site and individual.

Long-duration exploration missions will involve pro-longed exposure of astronauts to the microgravity ofspace, deceleration stresses, and intensive activity inthe partial-gravity environment of smaller planets andreturn to Earth. Management of the potentially serioushealth-care problem of bone loss may rely on clinicalchemical measurements. Clinical chemistry, performedduring space missions as part of a monitoring program,will be necessary to characterize the negative calcium

CLINICALCHEMISTRY, Vol. 39, No. 1, 1993 29

balance of individual astronauts during space missionsand to monitor the effectiveness of countermeasures forpreventing bone loss. Although the extent of osteopeniacaused by prolonged micro- or partial-gravity exposurehas not been determined, quantification of urinary cal-cium, which steadily increases during the first month ofmicrogravity exposure and levels off at about double thepreffight values, is of primary importance in detectingand monitoring the process (40-42). Urinary hydroxy-proline measurements may also be useful in monitoringincreased bone turnover (40). Most of the urine excre-tions have been measured in 24-h samples. However,calculating the calcium/creatinine and hydroxyproline/creatinine ratios from a 2-h urine collection may providea useful and more practical means of monitoring boneloss (47). Inflight total serum calcium concentrationsare higher than preffight values but are still within thenormal range (40-42). Better sensitivity to these minormetabolic disturbances might be observed with mea-surements of serum ionized calcium.

ElectrolyteTesting in Relationto Renal Stone Formation

Because of an increased knowledge of its pathophys-iological mechanisms and the development of new sur-gical therapies for it, nephrolithiasis is a disorder that isfairly well managed on Earth today (48). However,formation of a renal stone may have a greater probabil-ity as a medical problem in space, and could be moreserious and incapacitating. The loss of bone mass due tomicrogravity, such as that simulated by prolongedbedrest studies (49), would tend to produce hypercalci-uria and hyperphosphaturia, both of which are positiverisk factors for nephrolithiasis. In addition, moderatephysical exercise as a countermeasure to loss of bonemass and muscle strength, coupled with inadequatefluid intake due to suppression of thirst, could decreaseurine volume, further increasing the risk of renal-stoneformation (50). Surgical therapies, such as percutane-ous nephrostolithotomy and extracorporeal shock wavelithotripsy, probably will not be available in space.

Therefore, prevention and medical diagnosis and treat-ment appear to be the principal current options.

Implementation of a prevention program begins withan understanding of the known risk factors. Some ofthese are summarized in Table 2 (51). An extensivescreening program of astronaut candidates is also effec-tive in eliminating or treating those applicants whohave metabolic or environmental derangements predis-posing to nephrolithiasis (52). During long-durationmissions, effective countermeasures to include dietarycontrol together with a clinical chemical monitoringprogram may be the most effective ways to reduce therisk. At present, certain serum and urine chemicalanalyses are used to assess the potential for nephrolith-iasis, to confirm the diagnosis, and to monitor themedical treatment. The relevant clinical chemical as-says include serum and urine sodium, calcium, magne-sium, phosphorus and uric acid, and urine pH, volume,and relative density or osmolality (35). Oxalate, citrate,and sulfate analyses are available, but not as standard,automated tests incorporated in commercially availableanalyzers.

EnzymeTestingin Relationto SkeletalMuscleAtrophyLong-term absence of gravity causes skeletal muscle

atrophy if effective countermeasures are not used. Dur-ing Skylab, a negative nitrogen balance and a loss of 1.5kg of lean body mass was recorded for the crew mem-bers, with most of the loss occurring during the first fewweeks of flight (53). Increases in the concentration ofthyroxine and growth hormone have been implicated in

increased skeletal muscle turnover. Changes in the pre-and postifight serum and urine concentrations of creat-mine, sarcosine, 3-methylhistidine, and lactate dehy-drogenase isoenzyme during the Apollo-Soyuz TestProject (ASTP) indicated loss of skeletal muscle proteins(54). Recent Spacelab Life Sciences 1 experiments in-volved determination of the rate of synthesis and catab-olism by measurement of [‘5N]glycine uptake into pro-teins (55); results are pending.

Abnormality

MetabolicHypercalciunaHyperoxaluria

HypocitraturiaUrinepH <5.5UrinepH >7.0EnvironmentalLow urine outputHypematuriaHypersulfaturiaHyperphosphatemiaHypomagnesuriaPhysicochemical

Adaptedtroni Pak et al (51).

Table 2. RIsk Factors for Renal-Stone FormatlonMechanism

Saturated calcium oxalate and phosphateSaturated calcium oxalateDecreased calcium citrate complexes,

decreased inhibitor activityIncreasedundissociateduric acidAccentuated calcium phosphate saturation

Increasedsaturationof saltsIncreasedcalciumand calciumoxalateHypocitratunaSaturated calcium phosphateDecreased magnesium oxalate complexes

Supersaturation of calcium oxalate, brushite,sodium urate, struvite, uric acid

Possible cause

Excess absorption, bone lossIncreased absorptionor synthesis

Metabolic acidosisGouty diathesisRenal tubular acidosis

Inadequate fluidintakeExcesssodiumintakeHigh acid ash dietExcessdairy and meat intakeInadequateintakeof Mg


Surprisingly, ASTP results showed activities of totalcreatine kinase (CK) determined pre- and postmissionto be largely unchanged. A long-duration mission (e.g.,ASTP) without sufficient countermeasures might havebeen expected to cause a significant release of CK,similar to that found with human neurogenic atrophiesand dystrophies (56). Interpretation of CK and CK-MBisoenzyme analyses performed during a long-durationmission to confirm diagnosis of acute myocardial infarc-tion may require consideration of increased turnoverand repair of skeletal muscle tissue and specffic knowl-edge of the adaptation process for each crew member.Because of the increased skeletal muscle turnover dur-ing adaptation, CK would be expected to be muchgreater than C K-MB, and the relative activities wouldappear normal. However, an increased rate of produc-tion of CK-MB is also probable after damage and duringrepair of skeletal muscle. This adaptation has beenreported with neurogenic muscle atrophy (57) and afterstresses of long-distance running (58). Under thesecircumstances, increased concentrations of both CK andCK-MB in serum are expected, and the ability to con-firm a diagnosis of myocardial infarction by the crite-

rion of an increased CK-MB fraction of CK is compro-mised. Space adaptation may present a similar problem.

Endocrine Testing in Relation to Stress

The launch and the first few days of space travel areassociated with significant psychological and physicalstress. The complex endocrine responses to acute stressin humans are well documented and include release ofgrowth hormone, corticotropin, cortisol, the catechol-amines, /3-endorphins, and prolactin (59). Analyses ofblood and urine samples collected inflight and postffightduring Skylab and Shuttle missions document increasesin the concentrations of many of these hormones [seeFigure 2(31)].

Although automated immunoassays are now commer-cially available, operational analysis for hormones re-mains a low priority for SSF. All of the astronauts willhave undergone extensive preffight screening to detectthe presence of any underlying endocrine disorders.

However, for long-duration missions, the cumulativeexposure to radiation may have long-term detrimentaleffects, such as thyroid or pituitary damage, and mayrequire routine endocrine monitoring. This radiationexposure becomes especially important for space explo-ration missions beyond the currently planned low-Earth-orbit Shuttle series. Interpretation of hormoneconcentrations during missions must also account forchanges due to mission stresses. Urine collections for 24h and standard stimulation and suppression testingmay be necessary for accurate diagnosis during a mis-sion.

Interpretation of serum cortisol and corticotropin con-centrations would be particularly difficult for clinicaldiagnosis. Corticotropin and cortisol exhibit diurnalvariations, with morning values being higher thanevening values for most individuals. Recent animal andhuman studies show that cortisol secretion is more

linked with light and darkness cycles than with sleep-wake cycles (60-62). In SSF missions, assuming thatwindows are present to admit sunlight, “sunrise” willoccur every 90 mm. At the lunar surface, 14-day periods

of light and dark would alternate. During a Marsmission, sun exposure would be substantially less thanon Earth because of the greater distance from the sun.Control of light and dark conditions within a planetsurface habitat or space vehicle may be an importanthuman factor, the physiological effects of which could beeffectively monitored by analyses for corticotropin, cor-tisol, and related analytes (62).

Evaluation of Hepatic Function

Normal hepatic function in space and the potential ofmicrogravity for producing liver disease have not beenextensively studied. Preffight analyses for alkalinephosphatase, lactate dehydrogenase, and bilirubin inserum samples from Skylab astronauts have shown nosignificant differences from postffight results (32). Incontrast, pre- and postifight results of analyses of serumsamples from ASTP astronauts showed minor increasesin alkaline phosphatase, lactate dehydrogenase, andaspartate and alanine arninotransferases, but no changein bilirubin concentrations (63). Increases in lactatedehydrogenase were attributed to lung injury from aninadvertent release of toxic gases (mostly nitrogentetroxide) during landing. Loss of skeletal muscle andbone may account for release of the other enzymes.

Although there has been no history of significanthepatic disease related to space travel in the Americanspace program, a capability for standard hepatic enzymeanalyses is planned for the SSF HMF, including thoselisted above and y-glutamyltransferase, albumin, totalprotein, and direct bilirubin (26). These analyses wouldbe used to diagnose toxic, infectious, and traumatic liverinjury.

Pharmacokinetics and Therapeutic Drug Monitonng

Therapeutic drugs are used during Shuttle missionsfor treating space motion sickness (64). Unfortunately,the effects of microgravity on the pharmacokinetics ofthese or any other drugs have not been extensivelyexamined, largely because such studies require thecollection of many blood samples. Saliva samples havebeen collected to investigate absorption of acetami-nophen and scopolamine during Shuttle missions (27).The rate of absorption is decreased for these drugs,whereas the rate of elimination is unchanged. Furtherstudies with blood samples are needed because of thelimitations in the collection and interpretation of sali-vary drug concentrations (65). Blood collections for theevaluation of inifight pharmacokinetics of drugs arebeing planned for future missions (27). However, SSFcurrently has no plan for prospective therapeutic drugmonitoring during missions, although commonly moni-tored antibiotic and cardiovascular drugs are planned tobe part of the onboard HMF formulary (26). Becausemany clinical chemistry analyzers can measure theconcentrations of several therapeutic drugs, this capa-

Prellight Inflight Preflight Inhlight

Fig. 3. Comparison of D1H resultsfrom Shuttle missionsof threedifferentdurations:numberof positivereactions(left) andmeansumof reaction diameters (right)() 4 days,3crew members;(#{149}),5 days,3 crew members;() 10days,4 crewmembers. Source: Taylor and Janney (74); reprInted with permission


bility should be planned for if space medical technologyis to remain current.

ClInIcal ImmunologIcal AdaptatIons to Space

Alterations in the human immune mechanism, and inmicroorganisms potentially capable of effecting an infec-tious process, have been studied from the early days ofhuman spaceffight (66-69). Although the derived datahave often been incomplete, and sometimes ambiguous,one can draw several general conclusions. Some of theimmunological changes reported from the American andRussian studies are summarized in Table 3. These dataindicate that space flight can be expected to produce ablunting of the human cellular immune mechanismconcomitant with an increase in potentially pathogenicmicroorganisms, thereby increasing the probability ofmnfiight infectious diseases. During the early days of theAmerican space program, there was a very high inci-dence of infections before adoption of a preffight healthstabilization program. During the Apollo program, crewmembers experienced upper respiratory problems, influ-enza, viral gastroenteritis, rhinitis, pharyngitis, and milddermatological problems (68). Moreover, long-durationRussian space missions have been curtailed because ofoutbreaks of infectious disease (78).

Immunological Response to Spaceflight

The cellular immune response of American and Rus-sian crew members has been studied by various methodsfor >20 years. However, there remains a paucity ofreliable data for conclusions. Considerable immunolog-ical testing was performed after the Apollo, Skylab, andASTP missions (79). Also, postifight alterations in thein vitro response of cosmonaut lymphocytes were re-ported after Soyuz and Salyut (80, 81). However, be-cause of small sample size, mission anomalies, andconstraints on analytical conditions, the results weregenerally inconclusive (69).

More recently, extensive comparisons of pre- andpostmission immunological variables were conductedwith Shuttle astronauts (69, 71) and are summarized inTable 4. These comparisons demonstrated that the ab-solute number of lymphocytes, the ability of these cellsto respond to mitogenic stimulation, and the number ofeosinophils in the peripheral circulation were typicallydecreased after ffight. Conversely, there was an almostuniversal doubling of the absolute neutrophil number,and often a major change in the CD4/CD8 ratio. Thislatter event resulted from an increase in the helperlymphocyte population, as determined by flow-cytomet-ric analysis (71). Data from Shuttle missions 41B and41D, involving 11 crew members, indicated a postffightdecrease in circulating monocytes and B-lymphocytes.Further, the decreased T-lymphocyte blastogenesis cor-related with this decreased monocyte count. Becausemonocytes serve a critical role during lymphocyte acti-vation, acting as potent immunoregulator cells throughthe release of cytokines, these findings suggested apossible mechanism for the blunted in vitro mitogen-induced blastogenesis (79).

Only recently has the effect of spaceffight on theability of the human cell-mediated immune (CMI) sys-tem to function normally in vivo been tested inflight.This was accomplished by using the delayed-type hyper-sensitivity (DTH) response to common recall antigens asa simple yet effective method for evaluating inflight-mediated hypoergy in Shuttle crew members. The CMImechanism was evaluated in 10 astronauts by measur-ing their inflight DTH response to the common recallantigens of tetanus, diphtheria, Streptococcus, Proteus,old tuberculin, Candida, and Trichophyton. Both themean number of reactions and the aggregate reactionsize within this population were reduced inflight (Fig-ure 3). Further, the data suggest that on day 4 of aShuttle mission the CMI system was measurably de-graded, and that between days 5 and 10 the depressionwas maximized and the CMI mechanism began to adjustto the new conditions (74).

In future long-duration space flights, DTH should beused as an inflight screening test. This would simply yeteffectively demonstrate qualitative and quantitative invivo CMI dysfunction associated with spaceffight. Whenrelating DTH results to previous spaceffight immunol-ogy studies, it is important to remember that cells of themacrophage lineage are generally considered to be themain antigen-presenting cells in the DTH reaction (82).As has been previously shown, monocyte numbers in theperipheral circulation are altered postffight, the magni-tude of the depression being related to the decrease inmitogen-induced lymphocyte blast transformation (69,71). Because bloodborne macrophages and lymphocytesmake up the later phase of a normal DTH reaction (83),a connection between these events should be considered.

The presence of functioning delayed hypersensitivitycells is also required for a normal DTH response. Thesecells are the T lymphocytes that have become sensitizedto a particular antigen by a previous encounter (82). Analmost universal decrease in the number of T lympho-cytes in the peripheral circulation has been shown after

(I,C0

aacra>

00

az

70, 71

7271, 73

73

74

75

68

7677

Table 4. Summary of Posttllght Changes In Shuttle Crew Peripheral Blood CellsNo. of No. with No. with

sublects Increasedresults decreasedresults41 9 3141 5 3641 40 141 4 3511 6 511 4 711 3 711 8 311 5 511 7 4

Average

-13.3

-25.7+ 102.0

NA+ 1.6+9.7

-11.6+ 11.1

-2.3+ 13.4


Table 3. ImmunologIcal and MIcrobIologIcal ChangesReported durIng AmerIcan and Russian Spacefllght

Response RfersncssImmunologicalMajordepressionintheabilityof blastcells to

transform in responseto mitogenicchallengeLossof cytokine productionor functionMajor change In peripheral or splenicimmunecell

populationsAlterations in natural killer cell activityand

responseto colonystimulatingfactorDepressionin delayed-typehypersensitivity

responseMicrobiologicalSimplificationof crew autoflora

Reduction of saprophytesIncrease of potentially pathogenic

microorganismsBuildupof yeasts and filamentousfungiwithinthe

space cabinMicrobialcontaminationbetween crew membersIncreasedpathogenicityof certainspeciesafter

spaceflight

spaceffight (69, 71). However, whether or not this de-crease relates to a change in the functioning delayedhypersensitivity cells at the site of antigen applicationis not known. Data from humans are not available;however, increased numbers of T lymphocytes havebeen reported in the marrow of rats after short-termspaceflights (66, 84). It has been speculated that thenoted decrease of T lymphocytes in the peripheral cir-culation may be due to migration of T lymphocytes tothe bone marrow, as may occur in acute or chronic stressand in certain drug responses (66). If this theory iscorrect, such a migration could also result in a depletionof functioning delayed hypersensitivity cells or T helpercells, or both, at the site of antigen application.

When sensitized T cells are stimulated with the ap-propriate antigen, they undergo lymphoblastoid trans-

formation and cell division. This response provides ameasure of the function of the T cell memory, or T cellreproduction, or both (82). Again, there is an analogousdefect in peripheral blood. As has been demonstrated,starting with the first Shuttle flight, postifight mitogen-

mediated lymphocyte blast transformation may beblunted by as much as 60% from the subject’s preffightvalue (69, 71). In vitro blast transformation tests and invivo DTH tests evaluate complementary CMI systemcomponents (85). Therefore, to obtain a more completeunderstanding of the affected mechanism, these studiesshould be used in tandem for evaluating inflightchanges in the human CMI system.

In vivo T cell proliferation requires interferon gamma(INF-g) and probably interleukin 1 (IL-i) (82). In addi-tion, proper secretion and activity of interleukin 2 (1L-2)are necessary for feedback control between lymphocytes(83). Significant decreases in interleukin production(especially IL-2) and INF-g have been reported in cos-monauts and in rodents after spaceffights �168 days(66,80,81,86,87). A thorough inflight investigation ofT lymphocyte activity is essential for determining thedegree to which interleukins contribute to the identifiedCMI dysfi.mction. Such investigations should include,

but not be limited to, (a) IL-i-mediated activities such asprostaglandin production and activation of naturalkiller cells, macrophages, and lymphocytes; (b) the bal-ance between IL-2 receptor activity and IL-2 production;and (c) INF-g production by activated T lymphocytesand natural killer cells.

Finally, it will be important to analyze the inflightimmune system results in light of appropriate neuroen-docrine data and reliable estimates of the stress envi-romnent experienced by each crew member. In this way,the degree to which microgravity-induced changesthroughout the body influence the immune system, andvice versa, can be determined.

Microbiological Complications of a Blunted ImmuneResponse

Extensive microbiological studies were conductedaboard Apollo (88), Skylab (89), ASTP (76), and Soyuzand Mir (67) missions. On early Apollo missions, beforestrict protective measures were instituted, inflight in-fections were not unusual. However, preffight isolationwas initiated with the Apollo 14 mission as a counter-measure and remains an integral part of the Americanprogram (76). This practice, which allows the autoflorato equilibrate at an amount consistent with confInement

Factor

LymphocytenumberLymphocytestimulationNeutrophilnumberEosinophilpercentPan T lymphocytePan B lymphocytePan monocyteT helperT suppressorT4/T8 ratio

a Not useful because the count is typically reduced from a small number preflight to zero postflight.

Source: Taylor and Dardano (69).


and allows any infectious agents contracted to demon-strate themselves before flight, probably contributed tothe significant reduction in microbial problems duringand after space missions subsequent to Apollo 13 (75,78). Other patterns established early in the humanspaceflight experience were intercrew transfer of patho..gens (68), postflight increases in the number of sitescontaminated with such microorganisms as Staphylo-coccus aureus and f3-hemolytic streptococci (68), andincreased resistance of some microorganisms to antibi-otics (77).

A major concern during the pre-Shuttle days was thatupon return to Earth, crew members might respondnegatively to renewed contact with microorganismsthat were largely absent in the spacecraft. It wasthought that reduced exposure to microorganisms in-ffight would cause a decrease in both host immunocom-petence and the presence of competing microflora, whichcould result in a “microbial shock” upon return andexposure to a normal microbial load. However, samplesobtained immediately after landing showed no decreasein potentially pathogenic species. Therefore, the de-creased exposure to pathogens is not a factor in predict-ing inflight or postffight disease probabilities (76, 88,89).

Although inflight sampling has been limited, micro-biological specimens have been collected routinely be-fore and after Shuttle flights from the Shuttle air andinternal surfaces, the crew’s food, and the drinkingwater (90). Postifight orbiter air samples typically con-tain fungi (e.g., Aspergillus sp. and Penicillum sp.) andbacteria (e.g., Bacillus sp. and Micrococcus sp.). Addi-tionally, Staphylococcus sp., normally associated withskin, have been recovered in large numbers from theorbiter’s internal surfaces, but not from air samples(90).

Inflight sampling was conducted during the Spacelab3 mission because of concern about possible contamina-tion from the Research Animal Holding Facility (90,91). Sampling of air, holding-facility surfaces, and crewmembers’ throats and hands revealed an increase ofairborne fungi and bacteria within the laboratory (90);and fecal microorganisms, such as Escherichia coli andStreptococcus faecalis, were isolated from the interiorsurfaces of the facility postffight (91). However, in nocase was an extensive increase in the microbial loaddemonstrated.

Plans for microbiological analyses on future long-duration missions include provisions for inflight analy-sis of clinical and environmental samples to ensure thehealth, safety, and productivity of crew members (91).An inflight capability will exist to analyze crew, air,water, and spacecraft surface samples for microbialidentification and for determination of susceptibility toantimicrobial agents (91).

The effect of spaceflight on the infectious diseaseprocess must be closely monitored, and the ability toapply appropriate countermeasures must be maintained(91). Planners of long-duration missions must accommo-date inflight monitoring of microbial and immunologi-

cal variables, inflight analysis of samples and person-nel, and application of the countermeasures required tocounteract inifight immune dysfunctions and poten-tially harmful microbial imbalances.

Required Inflight Monitoring

The microbiological and immunological monitoringrequired consists of three different types of activities: (a)Certain testing must be done during the mission, eitherbecause the test is done on or in the body, because thesample is too labile to return to Earth, or because thedata must be known immediately for countermeasureadjustment. This component would include, but not belimited to, in vivo DTH evaluations and certain medicalmicrobiological tests, such as throat samples forf3-Streptococcus or total bacterial quantification inurine. (b) Other tests must be done on samples returnedto Earth, usually because the assay is too difficult to beperformed in the space environment, the equipment istoo complex or requires excessive resources for a planetsurface habitat or a space vehicle, or the procedurerequires a prohibitive amount of technician (crew) time.These tests would include all but the most basic micro-bial identifications and assays involving liquid chroma-tography, radioimmunoassay, or other methods. (c) Theremaining tests may be done either inflight or uponreturn of samples to Earth-the decision dependinglargely on the inflight availability of the assay orsample storage capability. This category may includecertain types of immune cell analyses and quantifica-tions, as well as identification of microorganisms otherthan expected frank pathogens. Both assay capabilityand sample storage (especially if refrigerated or frozen)are difficult to provide inflight. Therefore, the decision ofwhether to do these tests inflight or upon return will bedetermined by the available facilities.

Technology and DIrectIon for Space ExploratIon

The evolution of clinical laboratory technology hasbeen toward operator-independent, automated, high-throughput systems able to support laboratory require-ments of entire hospitals, medical centers, or geograph-ical regions. Automation of sample processing mayextend this trend and offer technology capable of beingused directly for space medicine applications. An exam-ple is the Integrated Blood-Collection System beingdeveloped by Kodak. This system makes use of a sophis-ticated container for sample collection, sample process-ing, metering of a discrete volume for analysis, andcontainment and disposal of excess sample volume (92).

These functions are likely to be important for laboratoryanalyses in space that require a combination of reagentsand sample fluid volume. Both critical care on Earthand medical care in space require efficient use of timeand sample volume and may benefit from this technol-ogy.

Technology for sophisticated, relatively low-through-put analyzers designed specifically for bedside or ICUapplications is likely to be most directly applicable toclinical space medicine. Developments of the 1980s


included physician’s office chemistry analyzers and low-maintenance, modular blood gas and electrolyte analyz-ers for critical-care applications (93). These develop-ments encouraged the initial developments for the SSF

HMF because these commercial instruments were seento have desirable characteristics for space medicineequipment (18). One of these analyzer systems, theReflotron” reflectance photometer (Boehringer Mann-heim GmbH, Vienna, Austria), modified for electricalpower supply aboard the Russian Space Station Mir,was tested by crew members on Mir during 1988-9, andremains onboard (JJ Un, General Electric Govt. Serv-ices, Inc., Houston, TX, personal communication, Janu-ary 1992). However, no reports of performance or ofresults of specific studies are at present available.

Stat or satellite laboratories, described as a matureconcept in 1981 (94), are now standard in US hospitals.In a 1989 survey of 33 hospitals in 19 states, all thosewith �450 beds used satellite laboratory facilities in ornear ICUs, emergency rooms, or operating rooms (19).Development of compact, reliable instrumentation forbedside laboratory diagnostics will likely continue thetrend from centralized laboratories and further encour-age the development of analyzer systems for spacemedicine applications. The combination of blood gas,chemistry, and hematology assays into a single compactinstrument for bedside use seems feasible and applica-ble to critical care in space or on Earth.

Certain analytes, if monitored continuously, couldprovide better diagnostic information than periodicmeasurement by laboratory analyzers. Currently avail-able transcutaneous technologies provide blood gas con-centrations and oxygen saturation. Developing technol-ogies may offer continuous monitoring of blood gases,glucose concentration, and certain electrolyte activitiesvia indwelling intravascular probes. Although continu-ous percutaneous intra-arterial monitoring of blood gasconcentrations, pH, and pressure addresses present in-tra-arterial catheter functions, the need for arterialplacement of the monitoring device would require ex-pertise similar to that required for catheter placement.The development of indwelling, continuous monitors ofall analytea currently used in laboratory diagnosis orclinical chemistry is unlikely in the near future, and invitro analysis of discrete fluid samples will most proba-bly remain standard for many years. However, single-or multiple-sensor probes, such as for blood gases, pH,electrolytes, and blood pressure, could quickly becomestandard technology (95, 96).

Clinical chemistry, immunology, and hematologyshould be components of space systems for clinicalmedicine and life science for long-duration missions.Laboratory diagnostic equipment for use in space mustdevelop as a part of the space medicine system for whichit is intended. The development process will requireactive, cooperative involvement of clinicians who uselaboratory information, laboratory scientists, biomedi-cal engineers, and aerospace engineers and managers.

References1. Garshnek V. Exploration of Mars: the human aspect. J BrInterplanetary Soc1990;43:475-88.2. Nicogossian AE. Overall physiological response to spaceflight.In: Nicogossian AE, Huntoon CL, Pool SL, eds. Space physiologyand medicine. Philadelphia, PA: Lea & Febiger, 1989:139-53.3. System integration plan for space shuttle extended durationorbiter program. NSTS 08306. Houston, TX: NASA, JohnsonSpace Center, June 1991.4. Extended duration orbiter medical project investigation re-quirements. Houston, TX: NASA, Johnson Space Center, MedicalSciences Division, July 1990.5. Medical operations requirements document for NSTS (Rev D).JSC.13956. Houston, TX: NASA, Johnson Space Center, Spaceand Life Sciences Directorate, Medical Sciences Division, June1990.6. Spaceshuttle program: flight data file: medical checklist (Ge-neric, Rev A). JSC-48031. Houston, TX: NASA, Johnson SpaceCenter, Mission Operations Directorate, Systems Division, Sep-tember 1990.7. Reportto Congress on the restructured Space Station Program.Washington, DC: NASA, Office of SpaceFlight, 1991.8. RobinsonJ. Restructure configuration convergence white pa-per: CHeCS (Rev B). Houston,TX: NASA, Johnson Space Center,SpaceStation Projects Office, February 1991.9. ACRV integrated operations concept document (Rev A). Hous-ton, TX: NASA, Johnson Space Center, Assured Crew ReturnVehicle Project Office, April 1991.10. America at the threshold: America’s space exploration initia-tive. Washington, DC:US Govt. Printing Office, 1990:114.11. International Mars mission. Proc., Toulouse, FR, session.Cambridge, MA: International Space University, 1991.12. McKinley BA, Mathes KL, Simmons SC. Preliminary designof health care systems for space exploration. SAE Tech Paper Ser911369. Warrendale, PA: SAE hit., 199l:l8pp.13. Houtchens BA. Final report: system for the management oftrauma and emergency surgery in space. Washington, DC: NatITechInf Service N84-2 1045, l984:l24pp.14. Gunby P, Ziporyn T, Marwick C, Merz B. Space medicine facesmassive task as humans venture farther from Earth. JAM.A1986;256:2009-52.15. Raymond CA. When medical help is really far away. JAMA1988;259:2343-4.16. Hamilton RJ. An operationally oriented approach to medicalcare in space. Aviat Space Environ Med 1991;62:1196-9.17. Tonnesen AS. Clinical chemistry laboratory requirements forthe critically ill or injured patient. Proc Annu hit Conf IEEE EngMed Biol Soc 1988;10:1795-6.18. Houtchens BA. Health-care systems for extended durationspaceflight. Clin Chem 1993;39:713-21.19. Fleisher MF, Schwartz MK. Strategies for organization andservice for the critical-care laboratory. Clin Chem 1990;36:1557-61.20. Hall JR. Critical-care medicine and the acute-care laboratory.Cliii Chem 1990;36(Suppl):1552-6.21. Bradshaw KE, Gardner RM, Clemmer TP, et al. Physiciandecision-making-evaluation of data used in a computerized ICU.Int J Clin Monit Comput 1984;1:81-91.22. Harris CE, MacMillan R, Atherton ST, Griffiths R. Retrospec-tive audit of out-of-hours laboratory tests in an intensive care unit.Clin Intensive Care 1991;2:200-3.23. Salem M, Chernow B, Burke R, et al. Bedside diagnostic bloodtesting: its accuracy, rapidity, and utility in blood conservation.JAMA 1991;266:382-9.24. McKinley BA. Development of a clinical chemistry analyzerfor medical laboratory diagnosis at the NASA International SpaceStation Freedom. Jpn J Clin Lab Autom 1990;15:1O-8.25. Wu AHB, Gonnet TG, Schenkel 0, Smith-Cronin L, GrahamGA, Tonnesen AS, McKinley BA. Preliminary evaluation of anexperimental clinical chemistry analyzer developed for space med-icine. Clin Chem 1993;39:37-44.26. Requirements of an in-flight crew health care system (CHeCS)for space station. JSC 31013 Rev C. Houston, TX: NASA, JohnsonSpace Center, Space Station Projects Office, March 1990.27. Huntoon CL, Johnson PC, Cintron NM. Hematology, immu-nology, endocrinology, and biochemistry. In: Nicogossian AR,

CLINICAL CHEMISTRY, Vol. 39, No. 1, 1993 35

Huntoon CL, Pool SL, eds.Spacephysiology and medicine. Phila-delphia, PA: Lea & Febiger, 1989:222-9.28. Phillips RW. S1.S-1. The first dedicated life sciences Shuttleflight. 63rd Ann Sci Mtg, Aerospace Med Assoc,Miami Beach, FL,May 1992.29. Cardiovascular and pulmonary systems. In: Committee onSpace Biology and Medicine, Space Science Board, eds. A strategyfor space biology and medical science. Washington, DC: NationalAcademyof Sciences Press, 1987:128-42.30. Marshall E. Space may be bad for your health [News). Science1991;253:1491.31. Leach CS. An overview of the endocrine and metabolicchanges in manned space flight. Acts Astronaut 1981;8:977-86.32. Leach CS, Rambaut PC. Biochemical responses of the Skylabcrewmen. In: Johnson RS, Dietlein LF, eds, Skylab Life Sci Symp.Houston, TX: NASA, Johnson Space Center, 1974:427-54.33. Newman AR. Spacelab Life Sciences I: living with micrograv-ity. Anal Chem 1990;62:1213-6A.34. Inaba H, Hirasawa H, Mizuguchi T. Serum osmolality gap inpostoperativepatients in intensive care. Lancet 1987;ii:1331-5.35. Penney MD, Walters G. Are osmolality measurements clini-cally useful? Ann Clin Biochem 1987;24:566-.71.36. Weil MH, Henning RJ, Pun VK. Colloid oncotic pressure:clinical significance. Crit Care Med 1979;7:113-6.37. Tavassoli M. Medical problems of space ffight. Am J Med1966;81:850-4.38. Robbins SL, Cotran RS, Kuniar V. The musculoskeletal sys-tem. In: Pathologicbasis of disease,3rd ed. Philadelphia, PA: WESaunders, 1984:1317-67.39. Rambaut PC, Leach CS, Whedon GD. A study of metabolicbalance in crewmembers of Skylab IV. Acta Astronaut 1979;6:1313-22.40. Leach CS, Rambaut PC. Biochemical responses of the Skylabcrewmen: an overview. In: Johnston RS, Dietlein LF, eds. Biomed-ical results from Skylab. NASA SP-3777. Washington, DC: NASA,Sci and Tech Inf Office, 1977:204-16.41. Lutwak L, Whedon GD, Lachance PA, et al. Mineral, electro-lyte, and nitrogen balance studies of the Gemini VII fourteen dayorbital spaceifight. J Clin Endocrinol 1969;29:1140-56.42. Leach CS, Alexander WC, Johnson PC. Endocrine, electrolyte,and fluid volume changes associated with Apollo missions. In:Johnston RS, Dietlein LF, Berry CA, eds. Biomedical results ofApollo. Washington, DC: NASA, 1975:163-84.43. Somjen D. Bone remodeling induced by physical stress isprostaglandin E2 mediated. Biochim Biophys Acts 1984;310:456-61.44. Leach CS, Rambaut PC. Biochemical observations of longduration manned orbital spaceffight. J Am Med Women’s Assoc1975;30:153-72.

45. VorobyovEl, Gazenko OG, Genin AM, Egorov AD. Medicalresults of Salyut-6 manned space flights. Aviat Space Environ Med1983;54(Suppl 1):S31-40.46. LeBlanc AD, Schneider VS, Evans HJ, et al. Bone mineral lossand recovery after 17 weeks of bed rest. J Bone Miner Res1990;5:843-50.47. van der Wiel HE, Lips P, Nauta J, et al. Biochemical param-eters of bone turnover during ten days of bed rest and subsequentmobilization. Bone Miner 1991;13:123-9.48. Preminger GM, Pak CYC. The practical evaluation and selec-tive medical management of nephrolithiasis. Sem Urol 1985;3:178-84.49. Hwang TIS, Hill K, Schneider V, Pak CYC. Effect of prolongedbedrest on the propensity for renal stone formation. J Clin Endo-crinol Metab 1988;66:209-12.50. Sakhaee K, Nigam S, Snell P, et al. Assessment of thepathogenetic role of physical exercise in renal stone formation. JClin Endocrinol Metab 1987;65:974-9.51. Pak CYC, Skurla C, Harvey J. Graphical display of urinaryrisk factors for renal stone formation. J Urol 1985;134:867-70.52. Pak CYC, Hill K, Cintron NM, Huntoon CL. Assessing appli-cants to the NASA flight program for their renal stone-formingpotential. Aviat Space Environ Med 1989;60:157-61.53. Leonard JI, Leach CS, Rambaut PC. Quantitation of tissueloss during prolonged space ifight. Am J Clin Nutr 1983;38:667-79.

54. Leach CS, Rambaut PC, DiFerrante N. Amunoaciduria inweightlessness. Acts Astronaut 1979;6:1323-33.55. Leach CS, Schneider HJ. Spacelab Life Sciences 1 and 2:scientific research objectives.Physiologist1987;30:26-9.56. Jockers-Vretou E, Vassilopoulos D. Serum creatine kunase Bsubunit levels in neurogemc atrophies. J Neurol 1985;232:236-9.57. Jockers-Vretou E, Vassilopoulos D. Skeletal muscle CK-Bactivity in neurogenic muscular atrophies. J Neurol 1989;236:284-7.58. Apple FS, Rogers MA, Casal DC, et al. Creatine kinase-MBisoenzyme adaptations in stressed human skeletal muscle of mar-athon runners. J Appl Physiol 1985;59:149-53.59. Rose EM. Psychoendocrinology. In: Wilson JD, Foster DW,eds. Textbook of endocrinology, 6th ed. Philadelphia, PA: Saun-ders, 1981:663-6.60. Minton JE, Davis DL, Stevenson JS. Contribution of thephotoperiod to circadian variations in serum cortisol and melato-mm in boars. Domest Anim Endocrinol 1989;6:177-81.61. Hansen T, Bratlid T, Lingjarde 0, Brenn T. Midwinter insom-nia in the subarctic region: evening levels of serum melatonin andcortisol and after treatment with bright artificial light. ActsPsychiatr Scand 1987;75:428-34.62. Czeisler CA, Johnson MP, Duffy JF, et al. Exposure to brightlight and darkness to treat physiologic maladaptation to nightwork. N Engl J Med 1990;322:1253-9.63. Leach CS. Biochemistry and endocrinology results. In: Nico-gossian AR, ed. The Apollo-Soyuz Test Project Medical Report.NASA Spec Pub 411. Washington, DC: NASA, 1977:87-100.64. Graybiel A. Prevention and treatment of space sickness inShuttle-orbital missions. Aviat SpaceEnviron Med 1979;50:171-6.65. Pippenger CE. Drug protein binding: an overview. In: MoyerTP, Boeckx RL, eds. Applied therapeutic drug monitoring, Vol. I:fundamentals. Washington, DC:Am Assoc Clin Chem, 1982:55-61.66. Konstantinova N. Manned space flights and the immunesystem. Short-term ifights. In: The immune system under extremeconditions: space immunology. Pnobl Kosm Biol 1988;59:104-24.67. Konstantinova IV. Manned space flights and the immunesystem. Long-term ffights. Probl Kosm Biol 1988;59:73-104.68. Taylor GR.Spacemicrobiology [Review]. Annu Rev Microbiol1974;28:121-37.69. Taylor GR, Dardano JR. Human cellular immune responsive-ness following space ffight. Aviat Space Environ Med 1983;54:S55-9.70. Cogoli A, Tschopp P. Lymphocyte reactivity during space-flight. Immunol Today 1985;6:1-4.71. Taylor GR, Neale LS, Dardano JR. Immunological analyses ofU.S. space shuttle cnewmembers. Aviat Space Environ Med 1986;57:213-7.72. Sonnenfeld G, Mandel AD, Konstantinova IV, et al. Effect ofspace ffight on immune response: bone marrow cell response tocolonary stimulating factor and leukocyte subsets. Physiologist1992;35(Suppl 1): S222-3.73. Sonnenfeld G, Mandel AD, Konstantinova IV, et al. Effect ofspace flight on levels and activity of immune cells. Aviat SpaceEnviron Med 1990;61:648-53.74. Taylor GR, Janney RP, In vivo testing confirms a blunting ofthe human cell-mediated immune mechanism during space flight.J Leukocyte Biol 1992;51:129-32.75. Carmichael C, Taylor GR. Evaluation of crew skin flora underconditions of a full quarantine lunar-exploration mission. Br JDermatol 1977;97:187-96.76. Taylor GR, Zaloguev SN. Medically important microorga-nisms recovered from Apollo-Soyuz Test Project (ASTP) crew-members. Life Sci Space Res 1977;15:207-12.77. Zaloguev SN, Shunkareva MM, Utkina TG. State of the au-tomicroflora of skin tissues and certain natural immunity indicesin the astronauts AG Nikolaev and VI Serast’ianov before andafter ffight. Kosm Biol Med 1970;4:54-9.78. Nicogossian AR, Garshnek V. Historical perspectives. In:Nicogossian AR, HuntoonCL, Pool SL, eds. Space physiology andmedicine. Philadelphia, PA: Lea & Febiger, 1989:41.79. Taylor GR. Cell anomalies associated with spaceflight condi-tions. In: Zouhair M, ed, Immunobiology of proteins and peptides,Vol. 4. New York: Plenum Press, 1987:259-71.80. Konatantinova N, Nefedov YG, Yeremin AV, et al. Immuno-


logical reactivity and prediction of allergic complications in thecrew of the second expedition of Salyut 4. Kosm Biol Aviko Med1978;12:15-29.81. Konstantinova N, Antropova YN, Legen’kov VI, ZazhireyVD. Study of reactivity of blood lymphoid cells in crew members ofthe Soyuz-6, Soyuz-7, and Soyuz-8 spaceships before and afterffight. Kosm Biol Med 1977;7:35-40.82. Roitt IM, BrostoffJ, Male DK. Immunology. New York: (lowerMedical Pub, 1989.83. Geczy CL. The role of lymphokines in delayed-type hypersen-sitivity reactions. Springer Semin Immunopathol 1984;7:321-46.84. Sonnenfeld G, Taylor GR. Effects of microgravity on theimmune system. SAE Tech Paper Ser 911515. Warrendale, PA:SAE International, 1991.85. Turk JL. Delayed hypersensitivity. In: Research monographsin immunology. New York: Elsevier/North-Holland, 1990.86. Berry WD, Murphy JD, Smith BA, et al. Effect of microgravitymodeling on interferon and interleukin responses in the rat. JInterferon Res 1991;11:243-9.87. Sonnenfeld G, Mandel AD, Konstantinova N, et al. Effects ofspace flight on levels and activity of immune cells. Aviat SpaceEnviron Med 1990;61:648-53.88. Taylor GR. Recovery of medically important micro-organismsfrom Apollo astronauts. Aerospace Med 1974;45:824-8.

89. Taylor GR, Graves RC, Brockett RM, et al. Skylab environ-mental and crew microbiology. In: Johnston RS, Dietlein LF, eds.Biomedical results from Skylab. NASA SP-3777. Washington, DC:NASA, Sci and Tech Inf Office, 1977:53-63.90. Pierson DL, McGinnis MR, Mishra SIC, Wogan CF. Microbi-ology on Space Station Freedom. Houston, TX: NASA Conf Pub3108, 1991.91. Gaiser KK, Groves TO, Henney MR, et al. Microbiologicalmonitoring during the Spacelab 3 mission [Abstract]. Aviat SpaceEnviron Med 1986;57:503.92. Columbus RL, Palmer HJ. The integrated blood-collectionsystem as a vehicle into complete clinical laboratory automation.Clin Chem 1991;37:1548-56.93. Zaloga GP. Evaluation of bedside testing options for thecritical care unit. Chest 1990;97:185S-90S.94. Weil MH, Michaels 5, Pun VK, et al. The stat laboratory:facilitating blood gas and biochemical measurements for the crit-ically ill and injured. Am J Clin Pathol 1981;76:34-42.95. McKinley BA, Houtchens BA, Janata J. In vivo applications ofISFET5: summary of current laboratory research and probableclinical detectors. Ion Sel Elect Rev 1984;6:173-208.96. Halbert SA. Intravascular monitoring: problems and promise.Clin Chem 1990;36:1581-4.

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