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Ultrastructural characteristics and behavior of previously frozen, glycerolyzed, and deglycerolyzed human red blood cells
Ci~vomoLonr Vol. 4, No. 4, 1968
U L T R A S T R U C T U R A L CHARACTERISTICS AND B E H A V I O R OF P R E V I O U S L Y FROZEN, GLYCEROLYZED, AND DEGLYCEROLYZED HUMAN R E D BLOOD CELLS ~ GWENDOI,YN J. STEWART AND H E N R I M. TURNER
Depe~rlmen.t of Biology, Boston Universily, Boslon, Massachusetts 02215 ]le(l blood cells preserved by slow freezing in high concentrations of glycerol have been studied extensively for their ability to function when used clinically." ~' ''' The in vivo survival as measured by chromium labeling :~'' was correlated with osmotic fragility and density distribution. However, the ultrastructure of these cells has received lit~tle attention. The present study was an outgrowth of an earlier study "'~ which indicated that the red cell has a doublelayered integument, the inner l~yer of which is contractile and responsible for both maintenance and transform:ttion of slmpe. In this study samples from several units of blood were examined for shape, for surface characteristics, and, after washing, for contraction of cell contents. ~'IATERIALS AND ~'II'Yl'HODS
AH of the blood used in this study came. from units of glycerolyzed, frozen blood from the Chelsea Naval Hospital (in collaboration with Dr. II~o!?er~ ~-aleri)° The cells were prepared for electron microscopic examination by two procedures. In the first procedure siliconized slides were covered with fresh cell-free plasma from ~ healthy individual. The excess plasma was washed off with Tyrode's solution, after which the glycerol3:zed blood was immediately placed on the wet surface and allowed to remain for a few seconds. The excess blood was then washed off with Tyrode's solution and the slide was flooded with phG~phate-buffered 1% osmium tetraoxide fixative. The fixative was !eft on for 30 min, then washed off with distilled water. The slides were dehydrated with an ascending alcohol series; shado.wed with platinum; cast with carbon; and covered with 1% Formvar. When the Formvar was thoroughly dry, the laminate film was stripped, Received December 7, 1967. *This work was supported by contract No. 0280-0001 from the Oifiee of NavM Research.
accgrding to the procedure described by Stewart.S. o In the second procedure a small alliquot of the blood was placed in ~ screw top tube with an excess (approximately 10 volumes of fix to 1 volume of blood) of fix and allowed to remain for approximately 2 hrs at 0°C, after which the red cells were sedimented, the fixative replaced with fresh fixative, and held at 4°C overnight. The cells were again sedimented, washed once with distilled water, and dehydrated with ascending alcohol series. From the 100% alcbhol the suspension was spread on a clean, unsiliconized glass slide, shadowed and cast with ca.rbon, covered with Fonnvar and stripped for examination. Both procedures give whole amounts of red cells rather than replicas. Cells from ~he same units were prepared while they were still in 40% glycerol and again after the glycerol had been removed by various procedures. t~ESULTS
The most startling observation was the shape of glycerolyzed red celJs. Instead of being biconcave discs (Fig. 1) they were biconvex discs in both slide-fixed and tube-fixed specimens (Figs. 2, 3, 4). The surfaces of slide-fixed specimens from some units were s m o o t h (Fig. 3) while those from other units were pock-marked (Fig. 4). Inst~mces of app~rent fusion between adjacent cells were common (Figs. 2, 3). These areas were easily distinguished from areas which exhibited close apposition but no fusion (Fig. 3). Upon removal of glycerol, many red cells that had been biconvex discs reverted to biconcave discs, while others became discs of rather uniform thickness, but none remained biconvex (Fig. 5). Apparent fusion in which the cells were in close proximity or in which they were separated by a distance of 2 to 5 tt were occasionally observed. :In inst~mces in which the 189
190
G . J . S T E W A R T A N D I-I. M. T U R N E R
FIG. 1. Normal fresh hum~m red The biconcavity and contraction of tics of such cells. Tile "ghost" m a y tents. There is a zone between the detectable hemoglobin. × 12,000.
cells, slide-fixed immediately after blood was drawn. contents from the periphery are consistent characterisbe seen a short distance from the electron-dense conedge of the electron-dense contents which is free of
ULTI~ASTI~UCTURE OF RE]:) BLOOD CELLS
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ULTRASTRUCTURE
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Ft(~. 4. Glycerolyzed red cells,, tube-fixed immediately after thawing. The cell surfaces in this specimen show numerous small pock-marks of various sizes. There is apparent fusion between [hin areas of adjacent membranes. .It appears that tile cells are being p u l l e d apart.
There is a teal- in tile integument of one cell which resembles a tear i n a pieCe lot thick asbestos. The rhumboidaI fragments in tlie background cannot be identified. There are depressions in the cell surfaces. ><,12,000.
193
194
G . J . S T E W A R T A N D I-t, M. TURNER,
co,I-lb were sepa.rated, I:,he distance was bridged by a long,, rather thin structure which seemed to be fuseci into each of the two cells, Cell,!' S which were fixed on slides or in tubes after removal of giyeerot showed five basic " . mrms with regard the eontc,~.~ , , ~ s of il~tern'd to the " ghost,s. " These forms were: i) biconcave cells which exhibited shrinkage of the electrondense eomI:onent,, ) ~ from the periplmrv of tile eel[; 2) bieonea.ve cells which exhibited no slirinkage from the periphery; 3) a uniformly dense disc with e0nsiderabte shrinkage of the electron-dense components from th e periphery; 4) a~ uniformly electron-dense disc with no shrinkage of components from tile periphery; and-5) knobby or tear-drop shaped cells. Some of the forms are shown in Figure 5. DlSCUSSlOX The reversible shape transitions produced by the addition and removal of glycerol must result from some influence of glycerol on the red cell integument or on hemoglobin. In view of the recent evidence for a, comractile inner layer of integument, it, seems more likely tha~ the shape changes result from a change in. the adenosinc triphosphate (ATP) level produced by glycerol in a manner similar to the action of glycerol on muscleY G13cerol-tIeated muscle fibers are free of ATP, b u t permeable to-the substance, and retain their act,omyosin st~ructure and ability to contract when placed in suitable medium .eontaining AT,P. :Relaxation beyond rest length of glycerol-treated muscle was no.t discussed by Szent-Oyorgyi; however, post-rigor relaxation occurs in dead. striated muscle after prolonged storage even under aseptic conditions at, refrigeration temperat.ures where putrefactionshould be absent? /~.eversible shape transitions have been produe.ed in fresh cells by Varying the concentration of ATP? Physically, the shape .transitions can be explained by a s s u m i n g - 1 ) t h a t the inner layer of t)e red cell-integument is ecruposed of a protein which ls capable of both eontra.ction a n d relaxation , depending, upon conditions, especially the level of ATP; and 2) ,that the layer is properly oriented and much thickened ,in the.' center. If the areas of concavity contain more contractile protein (i.e., a thicker layer), they would be capable of increasing in size more than the peripheral area, 1"
*
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¢
thus sl/owing the cell to transform from a biconcave to a biconvex disc. When the glycerol is removed, enough ATP is probably .present or quickly gen'ers, ted to provide sufficient energy for parti~l o,' complete recovery of shape through contraction of the inner layer. Though the behavior jus~ described is strongly indicativ~e of a contractible protein, the protein could not be organized into large units such as those found in muscle. Evident& ' ' indicates tlmt tiff.s: protein is in the form of short fibrils. Tlmre i:.~ suggestive evidence that these fibrils may have some sort of organization which is normally obscured by impregnation with hmnoglobin and which is easily destroyed by hypotonic solutions. This layer is also capable of contraction beyond the "normal" size when provokedb3, some unidentified means involved in handling. However, the ability to contract as well as maintain shape is lost by some cells during storage? In a cell population, the ability to contract from the periphery is lost, more frequently than the ability to retain shape, suggesting that the loss of cent r~ctility precedes rela,xation. With red cells from deglycerolyzed, previously frozen blood, stored blood, or native clotting blood, susceptibility to rupture by flow stress was a common characteristic of cells showing every form and degree of contractility; however, such rupture :and splitt, ing of hemoglobin has never been observed in fresh, anticoagulated blood." Thus, contractibility and mechanical strength may both be lost, but. independently. The significance of the pock-marking on some cell surfaces and fusion between cells remains obscure. Su~n~AaY When fresh red cells from a, healthy human were viewed in whole mount with the electron microscope, they %ere,biconcave discs with the contents contr~/cted away from t,he peripl~ery. Glyeerolyzed (40/o), . previously frozen red blood cells similarly examined were thin, biconvex discs: When these cells were deglycerolyzed with isotonic solutions, many reverted to bmoncax.e "' ; discs, ~ome ~ of which exhibited typical contraction from the periphery, but most of whictl exhibited no such contraction. Some became dense discs of rather uniform thick-
UI,TRASTRUCTURE OF RED BLOOD CELLS
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FIa. 5. Deglycerolyzed cells, slide-fixed immediately after washing. Most of the cells have assumed some degree of biconc~vity, varying fromsllght to.,pron0unced. A few show no evidence of biconcavity. One kn0bb,~; cell is-presen~, The:radiatir~g lines ,are wrinkles in the supporting film formed b..c,m~e oi" overloading of the film with cells. ×]2,000.
i
196
G . J . STEWART AND It. M. T U l l N E R
hess, while some exhibited contraction from lhe periphery but no concavity. An occasional knobby cell was observed. The same patterns of contraction have been observed in stored blood. The SUl:faccs of some cells were pockmarked while other were not. In some instances fusion between ;we cells appeared to have taken place. R EFERENCES 1. Bendall. J R. Post,mortem changes in muscle. In St,~eture and function, of muscle, vol. III, G. 1t. Bourme, ed., pp. 227-274. Publisher, City, 1960. 2. ttuggins, C. E. Frozen blood: theory and practice. J. A. M. A., 198: 941-944, 1965. 3. Huggins, C. E. Frozen blood. Ann. Surg., 15"0: 643-649, 1964. 4. Huggins, C. E., and Grove-Rasmuss~:m, M. Advances in blood preservation. Postgrad. Med., 27: 557-562, 1965. 5. Nakao, M., Nakao, T., Yamazo, S., and Yoshikawa, H. Adenosine triphosphate and shape of erythroeytes. J. Bioehcm. (Tokyo), /t9: 487-492, 1961. 6. Stewart, G. J. The red blood cell integument:
7.
8.
9.
10. 11.
12.
UltrastrucLu,'e 'rod behavio,'. Microvasc. I(es., i~1 press. StEwart, G. J. Ultrastructural characteristics of red cell integuments. Proceedings of First International Congress on Hemorrheology, in press. Stewari~. G. & An electron microscopic study of platelets and fibriu during plasma clotting. Program of the Electron Microscope Society of America (abstr.), Twenty-third Annual Meeting, 1965. Stewart, G., "rod Stewart, L. (Introduced by G. P. Fulton). Subcellulm" components of human platelets revealed in thre~ dimensional detail (.'tbstr.). Anat. Rec., 15~: 428, 1966. Szent-Gyorgyi, A. Free-energy relations "rod contraction of actomyosin. Biol. 13u11., 9(.;: 140-161, 1949. ¥aleri, C. R., McCallum, L. E., and Danon, ]). /~.elationships between in vivo ~mvival and (1) dcnsit, y distribution, (2) osmotic fragility of previously frozen, autologous, agglomerated, deglycerolized crythrocytes. Transfusion, 6: 554-564, 1966. Valeri, C. R., Mercado-L~Igo, I~., and ])anon, D. l:~.elationship I~e(.ween osmotic fragility and.#~ vivo survival of lul[ologous deglyeerolized resuspended r(~d Mood cells. Transfusion, 5: 267-272, 1965.
U L T R A S T R U C T U R A L CHARACTERISTICS AND B E H A V I O R OF P R E V I O U S L Y FROZEN, GLYCEROLYZED, AND DEGLYCEROLYZED HUMAN R E D BLOOD CELLS ~ GWENDOI,YN J. STEWART AND H E N R I M. TURNER
Depe~rlmen.t of Biology, Boston Universily, Boslon, Massachusetts 02215 ]le(l blood cells preserved by slow freezing in high concentrations of glycerol have been studied extensively for their ability to function when used clinically." ~' ''' The in vivo survival as measured by chromium labeling :~'' was correlated with osmotic fragility and density distribution. However, the ultrastructure of these cells has received lit~tle attention. The present study was an outgrowth of an earlier study "'~ which indicated that the red cell has a doublelayered integument, the inner l~yer of which is contractile and responsible for both maintenance and transform:ttion of slmpe. In this study samples from several units of blood were examined for shape, for surface characteristics, and, after washing, for contraction of cell contents. ~'IATERIALS AND ~'II'Yl'HODS
AH of the blood used in this study came. from units of glycerolyzed, frozen blood from the Chelsea Naval Hospital (in collaboration with Dr. II~o!?er~ ~-aleri)° The cells were prepared for electron microscopic examination by two procedures. In the first procedure siliconized slides were covered with fresh cell-free plasma from ~ healthy individual. The excess plasma was washed off with Tyrode's solution, after which the glycerol3:zed blood was immediately placed on the wet surface and allowed to remain for a few seconds. The excess blood was then washed off with Tyrode's solution and the slide was flooded with phG~phate-buffered 1% osmium tetraoxide fixative. The fixative was !eft on for 30 min, then washed off with distilled water. The slides were dehydrated with an ascending alcohol series; shado.wed with platinum; cast with carbon; and covered with 1% Formvar. When the Formvar was thoroughly dry, the laminate film was stripped, Received December 7, 1967. *This work was supported by contract No. 0280-0001 from the Oifiee of NavM Research.
accgrding to the procedure described by Stewart.S. o In the second procedure a small alliquot of the blood was placed in ~ screw top tube with an excess (approximately 10 volumes of fix to 1 volume of blood) of fix and allowed to remain for approximately 2 hrs at 0°C, after which the red cells were sedimented, the fixative replaced with fresh fixative, and held at 4°C overnight. The cells were again sedimented, washed once with distilled water, and dehydrated with ascending alcohol series. From the 100% alcbhol the suspension was spread on a clean, unsiliconized glass slide, shadowed and cast with ca.rbon, covered with Fonnvar and stripped for examination. Both procedures give whole amounts of red cells rather than replicas. Cells from ~he same units were prepared while they were still in 40% glycerol and again after the glycerol had been removed by various procedures. t~ESULTS
The most startling observation was the shape of glycerolyzed red celJs. Instead of being biconcave discs (Fig. 1) they were biconvex discs in both slide-fixed and tube-fixed specimens (Figs. 2, 3, 4). The surfaces of slide-fixed specimens from some units were s m o o t h (Fig. 3) while those from other units were pock-marked (Fig. 4). Inst~mces of app~rent fusion between adjacent cells were common (Figs. 2, 3). These areas were easily distinguished from areas which exhibited close apposition but no fusion (Fig. 3). Upon removal of glycerol, many red cells that had been biconvex discs reverted to biconcave discs, while others became discs of rather uniform thickness, but none remained biconvex (Fig. 5). Apparent fusion in which the cells were in close proximity or in which they were separated by a distance of 2 to 5 tt were occasionally observed. :In inst~mces in which the 189
190
G . J . S T E W A R T A N D I-I. M. T U R N E R
FIG. 1. Normal fresh hum~m red The biconcavity and contraction of tics of such cells. Tile "ghost" m a y tents. There is a zone between the detectable hemoglobin. × 12,000.
cells, slide-fixed immediately after blood was drawn. contents from the periphery are consistent characterisbe seen a short distance from the electron-dense conedge of the electron-dense contents which is free of
ULTI~ASTI~UCTURE OF RE]:) BLOOD CELLS
191
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Fro. 2. Glycerolyzed red cells, slide-fixed immediately after thawing. The cells are thin, convex discs with what appears to be an area of fusion between the. two cells. The extxeme thinness is indicated by short shadow. Much amorphous debris is present in the background. X 12,000.
•
:
"O00'gt × "snouo2omoq pu~ avinuva~ ~qout3 aa~ gi[oa oaaq'~ [i v jo ~aa~jan~ mkL "UO.TSnJ jo aau~a'aadd~ m:I1 2 u [ , ~ 'po~aa~!iqo .~qo~IdmOa s.~ siioa o,'at uoa.~,,.',4oq uot]aun.¢ ami~ou~ 9I!q.v._. a~p!./ Ti'ams ~ s~ ~luapt,',,.o XIar.ala st o,~. a.~q~o m4~ q~!,v,. shoo oq~ lo auo Xq ap~m ~uo!~aunr OtkL "Siiaa paxg-op.[[~ oti?. m;q~ aauu!tI1 pu~ aopp,t oq o:l a~odd~ Slpa osml& "~u.~at~,q'l aa:Ij~ Xial,a.~pamm.~ paxg-aqn~.'Sllaa paa pazXlOaaaXIO "g " u ~
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ULTRASTRUCTURE
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OF
RED
BLOOD
CELLS
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Ft(~. 4. Glycerolyzed red cells,, tube-fixed immediately after thawing. The cell surfaces in this specimen show numerous small pock-marks of various sizes. There is apparent fusion between [hin areas of adjacent membranes. .It appears that tile cells are being p u l l e d apart.
There is a teal- in tile integument of one cell which resembles a tear i n a pieCe lot thick asbestos. The rhumboidaI fragments in tlie background cannot be identified. There are depressions in the cell surfaces. ><,12,000.
193
194
G . J . S T E W A R T A N D I-t, M. TURNER,
co,I-lb were sepa.rated, I:,he distance was bridged by a long,, rather thin structure which seemed to be fuseci into each of the two cells, Cell,!' S which were fixed on slides or in tubes after removal of giyeerot showed five basic " . mrms with regard the eontc,~.~ , , ~ s of il~tern'd to the " ghost,s. " These forms were: i) biconcave cells which exhibited shrinkage of the electrondense eomI:onent,, ) ~ from the periplmrv of tile eel[; 2) bieonea.ve cells which exhibited no slirinkage from the periphery; 3) a uniformly dense disc with e0nsiderabte shrinkage of the electron-dense components from th e periphery; 4) a~ uniformly electron-dense disc with no shrinkage of components from tile periphery; and-5) knobby or tear-drop shaped cells. Some of the forms are shown in Figure 5. DlSCUSSlOX The reversible shape transitions produced by the addition and removal of glycerol must result from some influence of glycerol on the red cell integument or on hemoglobin. In view of the recent evidence for a, comractile inner layer of integument, it, seems more likely tha~ the shape changes result from a change in. the adenosinc triphosphate (ATP) level produced by glycerol in a manner similar to the action of glycerol on muscleY G13cerol-tIeated muscle fibers are free of ATP, b u t permeable to-the substance, and retain their act,omyosin st~ructure and ability to contract when placed in suitable medium .eontaining AT,P. :Relaxation beyond rest length of glycerol-treated muscle was no.t discussed by Szent-Oyorgyi; however, post-rigor relaxation occurs in dead. striated muscle after prolonged storage even under aseptic conditions at, refrigeration temperat.ures where putrefactionshould be absent? /~.eversible shape transitions have been produe.ed in fresh cells by Varying the concentration of ATP? Physically, the shape .transitions can be explained by a s s u m i n g - 1 ) t h a t the inner layer of t)e red cell-integument is ecruposed of a protein which ls capable of both eontra.ction a n d relaxation , depending, upon conditions, especially the level of ATP; and 2) ,that the layer is properly oriented and much thickened ,in the.' center. If the areas of concavity contain more contractile protein (i.e., a thicker layer), they would be capable of increasing in size more than the peripheral area, 1"
*
'
•
¢
thus sl/owing the cell to transform from a biconcave to a biconvex disc. When the glycerol is removed, enough ATP is probably .present or quickly gen'ers, ted to provide sufficient energy for parti~l o,' complete recovery of shape through contraction of the inner layer. Though the behavior jus~ described is strongly indicativ~e of a contractible protein, the protein could not be organized into large units such as those found in muscle. Evident& ' ' indicates tlmt tiff.s: protein is in the form of short fibrils. Tlmre i:.~ suggestive evidence that these fibrils may have some sort of organization which is normally obscured by impregnation with hmnoglobin and which is easily destroyed by hypotonic solutions. This layer is also capable of contraction beyond the "normal" size when provokedb3, some unidentified means involved in handling. However, the ability to contract as well as maintain shape is lost by some cells during storage? In a cell population, the ability to contract from the periphery is lost, more frequently than the ability to retain shape, suggesting that the loss of cent r~ctility precedes rela,xation. With red cells from deglycerolyzed, previously frozen blood, stored blood, or native clotting blood, susceptibility to rupture by flow stress was a common characteristic of cells showing every form and degree of contractility; however, such rupture :and splitt, ing of hemoglobin has never been observed in fresh, anticoagulated blood." Thus, contractibility and mechanical strength may both be lost, but. independently. The significance of the pock-marking on some cell surfaces and fusion between cells remains obscure. Su~n~AaY When fresh red cells from a, healthy human were viewed in whole mount with the electron microscope, they %ere,biconcave discs with the contents contr~/cted away from t,he peripl~ery. Glyeerolyzed (40/o), . previously frozen red blood cells similarly examined were thin, biconvex discs: When these cells were deglycerolyzed with isotonic solutions, many reverted to bmoncax.e "' ; discs, ~ome ~ of which exhibited typical contraction from the periphery, but most of whictl exhibited no such contraction. Some became dense discs of rather uniform thick-
UI,TRASTRUCTURE OF RED BLOOD CELLS
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FIa. 5. Deglycerolyzed cells, slide-fixed immediately after washing. Most of the cells have assumed some degree of biconc~vity, varying fromsllght to.,pron0unced. A few show no evidence of biconcavity. One kn0bb,~; cell is-presen~, The:radiatir~g lines ,are wrinkles in the supporting film formed b..c,m~e oi" overloading of the film with cells. ×]2,000.
i
196
G . J . STEWART AND It. M. T U l l N E R
hess, while some exhibited contraction from lhe periphery but no concavity. An occasional knobby cell was observed. The same patterns of contraction have been observed in stored blood. The SUl:faccs of some cells were pockmarked while other were not. In some instances fusion between ;we cells appeared to have taken place. R EFERENCES 1. Bendall. J R. Post,mortem changes in muscle. In St,~eture and function, of muscle, vol. III, G. 1t. Bourme, ed., pp. 227-274. Publisher, City, 1960. 2. ttuggins, C. E. Frozen blood: theory and practice. J. A. M. A., 198: 941-944, 1965. 3. Huggins, C. E. Frozen blood. Ann. Surg., 15"0: 643-649, 1964. 4. Huggins, C. E., and Grove-Rasmuss~:m, M. Advances in blood preservation. Postgrad. Med., 27: 557-562, 1965. 5. Nakao, M., Nakao, T., Yamazo, S., and Yoshikawa, H. Adenosine triphosphate and shape of erythroeytes. J. Bioehcm. (Tokyo), /t9: 487-492, 1961. 6. Stewart, G. J. The red blood cell integument:
7.
8.
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