Comp. Biochem. Physiol., 1975, VoL 51A, pp. 275 to 280. Pergamon Press. Printed in Great Britain
COMPARATIVE HEMATOLOGY: STUDIES ON OPOSSUMS
DIDELPHIS MARSUPIALIS ( VIRGINIANUS)* JESSICA H. LEWIS Department of Medicine, University of Pittsburgh, Pittsburgh, Pennsylvania 15213, U.S.A. (Received 19 March 1974)
Abstract--The studies compared opossum to human blood. 1. Coagulation mechanisms appeared to be fundamentally similar although factors II, VII and X assayed well below and V, VIII, IX, XI and XII above the human range. Fibrinogen appeared to be quantitatively similar to human but different in its poor clottability with bovine thrombin and inability to clump staphylococci. 2. Platelets were variable in number and size and adhered to glass and aggregated with ADP much as do human platelets. On the other hand, they aggregated poorly or not at all with collagen, epinephrine and thrombin. 3. Platelet ultrastructure was similar to human except for two striking differences. Many platelets contained a circumscribed mass of wavy fine fibrillar material of unknown composition. Also, striated elongated granules were quite common. 4. Erythrocytes were slightly smaller than human. Leukocytes were not very different and lymphocytes predominated. 5. Serum albumin was very low by the automated dye-binding method but was much higher by electrophoresis. 6. The serum enzyme values were very much higher than those seen in normal humans.
INTRODUCTION THE OPOSSUM, Didelphis marsupialis (virginianus), is the only m a m m a l o f order Marsupialis found in N o r t h America. T w o other species live in South America and all of the rest of the living marsupials are confined to Australia. Hematological studies on Marsupials are sparse. Waring et aL (1966) summarized some pre-1965 publications. Blood coagulation studies have been made by Fantl (1957, 1961), Didisheim et al. (1959), Lewis et al. (1968), Parsons et al. (1970, 1971) and Parsons & Guiler (1972). Additional hematological and/or biochemical data concerning N o r t h American opossums are to be found in Youatt et aL (1961), Mays & Loew (1968), T i m m o n s & Marques (1969) and Giacometti et aL (1972). Rothstein & Hunsaker (1972) studied the South American woolly opossum. The studies reported here are part of a comprehensive, comparative study of the blood of vertebrates. A number o f new observations are reported. Striking differences f r o m h u m a n values were found in serum enzymes and in platelet ultrastructure. MATERIALS AND METHODS The opossums were trapped uninjured wild animals or were purchased from a biological supply house. In either * This study was supported by Grant HL-02254 from the National Institutes of Health.
case they were kept for a few days and fed raw eggs and vegetables. Blood was obtained by cardiac puncture. Coagulation, biochemical and hematological tests employed standard or previously published (Lewis, 1961, 1971) methods. For the electron microscope buffy coat platelet studies 9 ml blood was added to I ml 0.1 M sodium citrate at 37°C and promptly centrifuged at room temperature for 5 min at 2000 rev/min. The plasma was removed and 3 ml of freshly diluted warm 3% glutaraldehyde in 0.2 M phosphate buffer pH 7.4 was layered over the buffy coat. After 30 mill the glutaraldehyde was removed and the fixed burly coat separated and cut into 8 small thin slices. The slices were transferred to a fresh tube and rinsed with about ten changes of buffer during 30 min, then drained and 5 ml 1% osmic acid added. After 1 h the osmic acid was removed and the material dehydrated for two 10 rain periods in each concentration--50, 75, 90 and 100% ethanol. Propylene oxide was added for two 10-min periods followed by a 2 : 1 mixture of propylene oxide and Epon-Araldite. After 1 hr this was replaced by a 1 : 2 mixture of the same chemicals. The buffy coat slices stayed in this mixture for 8 or more hours and were then transferred to pure Epon and embedded using a fiat Teflon embedding block. The slices were oriented so that the cut end of a slice extended to the neck of the block. A Phillips 200 electron microscope was employed. Platelets were sized on a Wright stained smear with a calibrated eyepiece at 1000 × magnification. All of the chemical studies except HBD (hydroxybutyric dehydrogenase), CPK (creatine phosphokinase), LDH-L (lactic dehydrogenase) and SGOT (serum glutamic oxalacetic transaminase) were performed using
275
276
JESSICA H. LEWIS
400 >400 339 68
range 6.0-8.0 3"5-5-0 8.5-10"5 2.5-4.5 150-300 65-110 2.5-8.0 0-7-1.4 0.1-1.4 0-1-0.5 10-20 95-105 24-32 3.5-5-0 135-145 30-85 14-485 0-110 110-230 10-50 7-40
280
JESSICA H. LEWIS
serum. Frequently the a2 and /3 globulins merged with the y globulin into a large, gradually sloped curve in which individual proteins could not be differentiated. In two of the animals it was possible to distinguish peaks corresponding to those found in human serum. Biochemical tests (Table 8)
Striking findings are the apparently low levels of albumin and the high levels of serum enzymes. The latter are specific assays and the values should reflect true activity yet they are very much higher than those found in humans. Miscellaneous
Opossum fibrinogen did not clump a standard preparation of killed staphylococci (Newman D~C coagulase negative) used in the staphylococcal clumping test (Lewis & Wilson, 1973). Fibrinolytic activity was not found in the opossum euglobulin fraction in the presence of streptokinase in a final concentration of 20 or 200/z/ml. Urokinase (100/z/ml) appeared to have a weak activating effect. Bleeding times done on the underbelly skin were less than 1 rain. DISCUSSION Although many of the values observed in the clotting tests differed greatly from those found with human plasma this probably does not reflect any fundamental difference in the coagulation mechanisms. The long times observed with Simplastin® correlate well with the low values in the assays for II, VII and X and could be explained on the lack of activity with this thromboplastin, but it is difficult to understand the high value in the V assay which also employs Simplastin®. The PTT is faster than human, indicating an effective intrinsic mechanism, a finding supported by the T G T test. Platelets showed poor aggregation with collagen, epinephrine and bovine thrombin. This last observation together with the long thrombin time suggests that bovine and opossum materials interreact poorly. That opossum fibrinogen may differ from that of most mammals (Lewis & Wilson, 1973) is supported by the observation of lack of staphylococcal clumping as well as by the long thrombin time. Electron microscopic studies show many ultrastructural characteristics similar to those found in human platelets (White, 1972). About 10~ of the platelets contained fine curly fibrillar material whose character and function are unknown at this time. Acknowledgements--The author wishes to thank Dr. R. Iammarino for advice and for performing the serum electrophoretic and biochemical tests in the clinical laboratories of Presbyterian University Hospital. The author also wishes to thank Ms. Geraldine Morici for her beautiful electron microscope preparations and Ms. Mary Vogel and Messrs. George Arvay, Willie Cooks, James
Donohoe and John Wilson for performing the coagulation studies. REFERENCES DIDISHEIM P., HATFORI K. & LEWIS J. H. (1959) Hematologic and coagulation studies in various animal species, d. Lab. clin. Med. 53, 866-875. FANTLP. (1961) Comparative study of blood coagulation in vertebrates. Austral J. exp. Biol. Med. Sci. 39, 403-412. FANTL P. & WARD H. A. (1957) Comparison of blood clotting in marsupials and man. Austral. J. exp. Biol. Med. Sci. 35, 209-224. GIACOMETTIL., BERNTZENA. & BLISSM. (1972) Hematologic parameters of the opossum (Didelphis virginiana). Comp. Biochem. PhysioL 43A, 287-292. LEWIS J. H. (1961) Coagulation defects. JAMA 178, 1014-1020. LEWIS J. H. (1971) Hemostasis and hemorrhage. Sci. Clin. 1, 1-66. LEWIs J. H. (1974) Comparative hematology: studies on elephants Elephas maximus. Comp. Biochem. PhysioL 49A, 175-181. LEWIs J. H., PHILLIPSL. L. & HANN C. (1968) Coagulation defects in primitive Australian mammals. Comp. Biochem. Physiol. 25, 1129-1135. LEWISJ. H. & WILSONJ. H. (1973) Variations in abilities of animal fibrinogens to clump staphylococci, Thrombosis Res. 3, 419-424. MArS A. & LoEw F. W. (1968) Hemograms of laboratory-confined opossums (Didelphis virginiana). J.A.V.M.A. 153, 800-802. PARSONSR. S. & GUtLER E. R. (1972) Observations on the blood of the marsupial tiger cat, Dasyurops maculatus (Kerr) (Dasyuridae). Comp. Biochem. Physiol. 43A, 935-939. PARSONSR. S., GUILERE. R. & HEDOLER. W. L. (1971) Comparative studies on the blood of monotremes and marsupials--II. Electrolyte, organic constituents, proteins, gas analysis and enzymes. Comp. Biochem. Physiol. 39B, 209-217. PARSONSR. S., HEDDLER. W. L., FLUXW. G. & GUILER E. R. (1970) Studies on the blood of the Tasmanian devil. Comp. Biochem. Physiol. 32, 345-351. ROTHSTEIN R. & HUNSAKERD. (1972) Baseline hematology and blood chemistry of the South American woolly opossum, Caluromys derbianus. Lab. Anita. Sci. 22, 227-232. TIMMONSE. H. & MARQUESP. A. (1969) Blood chemical and hematological studies in the laboratory-confined, unanesthetized opossum, Didelphis virginiana. Lab. Anim. Care 19, 342-344. WARINGH., MOIR R. J. & TYNDALE-BISCOEC. H. (1966) Comparative physiology of marsupials. In Advances in Comparative Physiology and Biochemistry (Edited by LOWENSTEINO.), Vol. 2, pp. 237-376. Department of Zoology and Comparative Physiology, University of Birmingham. WHrrE J. G. (1972) Ultrastructural defects in congenital disorders of platelet function. Ann. N.Y. Acad. Sci. 201, 205-233. YOUATTW. G., FAY L. D., HOWE D. L. & HARTE H. D. (1961) Hematologic data on some small mammals. Blood 18, 758-763. Key Word lndex---Comparative Hematology; opossum; Didelphis marsupialis; platelets; blood coagulation.
COMPARATIVE HEMATOLOGY: STUDIES ON OPOSSUMS
DIDELPHIS MARSUPIALIS ( VIRGINIANUS)* JESSICA H. LEWIS Department of Medicine, University of Pittsburgh, Pittsburgh, Pennsylvania 15213, U.S.A. (Received 19 March 1974)
Abstract--The studies compared opossum to human blood. 1. Coagulation mechanisms appeared to be fundamentally similar although factors II, VII and X assayed well below and V, VIII, IX, XI and XII above the human range. Fibrinogen appeared to be quantitatively similar to human but different in its poor clottability with bovine thrombin and inability to clump staphylococci. 2. Platelets were variable in number and size and adhered to glass and aggregated with ADP much as do human platelets. On the other hand, they aggregated poorly or not at all with collagen, epinephrine and thrombin. 3. Platelet ultrastructure was similar to human except for two striking differences. Many platelets contained a circumscribed mass of wavy fine fibrillar material of unknown composition. Also, striated elongated granules were quite common. 4. Erythrocytes were slightly smaller than human. Leukocytes were not very different and lymphocytes predominated. 5. Serum albumin was very low by the automated dye-binding method but was much higher by electrophoresis. 6. The serum enzyme values were very much higher than those seen in normal humans.
INTRODUCTION THE OPOSSUM, Didelphis marsupialis (virginianus), is the only m a m m a l o f order Marsupialis found in N o r t h America. T w o other species live in South America and all of the rest of the living marsupials are confined to Australia. Hematological studies on Marsupials are sparse. Waring et aL (1966) summarized some pre-1965 publications. Blood coagulation studies have been made by Fantl (1957, 1961), Didisheim et al. (1959), Lewis et al. (1968), Parsons et al. (1970, 1971) and Parsons & Guiler (1972). Additional hematological and/or biochemical data concerning N o r t h American opossums are to be found in Youatt et aL (1961), Mays & Loew (1968), T i m m o n s & Marques (1969) and Giacometti et aL (1972). Rothstein & Hunsaker (1972) studied the South American woolly opossum. The studies reported here are part of a comprehensive, comparative study of the blood of vertebrates. A number o f new observations are reported. Striking differences f r o m h u m a n values were found in serum enzymes and in platelet ultrastructure. MATERIALS AND METHODS The opossums were trapped uninjured wild animals or were purchased from a biological supply house. In either * This study was supported by Grant HL-02254 from the National Institutes of Health.
case they were kept for a few days and fed raw eggs and vegetables. Blood was obtained by cardiac puncture. Coagulation, biochemical and hematological tests employed standard or previously published (Lewis, 1961, 1971) methods. For the electron microscope buffy coat platelet studies 9 ml blood was added to I ml 0.1 M sodium citrate at 37°C and promptly centrifuged at room temperature for 5 min at 2000 rev/min. The plasma was removed and 3 ml of freshly diluted warm 3% glutaraldehyde in 0.2 M phosphate buffer pH 7.4 was layered over the buffy coat. After 30 mill the glutaraldehyde was removed and the fixed burly coat separated and cut into 8 small thin slices. The slices were transferred to a fresh tube and rinsed with about ten changes of buffer during 30 min, then drained and 5 ml 1% osmic acid added. After 1 h the osmic acid was removed and the material dehydrated for two 10 rain periods in each concentration--50, 75, 90 and 100% ethanol. Propylene oxide was added for two 10-min periods followed by a 2 : 1 mixture of propylene oxide and Epon-Araldite. After 1 hr this was replaced by a 1 : 2 mixture of the same chemicals. The buffy coat slices stayed in this mixture for 8 or more hours and were then transferred to pure Epon and embedded using a fiat Teflon embedding block. The slices were oriented so that the cut end of a slice extended to the neck of the block. A Phillips 200 electron microscope was employed. Platelets were sized on a Wright stained smear with a calibrated eyepiece at 1000 × magnification. All of the chemical studies except HBD (hydroxybutyric dehydrogenase), CPK (creatine phosphokinase), LDH-L (lactic dehydrogenase) and SGOT (serum glutamic oxalacetic transaminase) were performed using
275
276
JESSICA H. LEWIS
400 >400 339 68
range 6.0-8.0 3"5-5-0 8.5-10"5 2.5-4.5 150-300 65-110 2.5-8.0 0-7-1.4 0.1-1.4 0-1-0.5 10-20 95-105 24-32 3.5-5-0 135-145 30-85 14-485 0-110 110-230 10-50 7-40
280
JESSICA H. LEWIS
serum. Frequently the a2 and /3 globulins merged with the y globulin into a large, gradually sloped curve in which individual proteins could not be differentiated. In two of the animals it was possible to distinguish peaks corresponding to those found in human serum. Biochemical tests (Table 8)
Striking findings are the apparently low levels of albumin and the high levels of serum enzymes. The latter are specific assays and the values should reflect true activity yet they are very much higher than those found in humans. Miscellaneous
Opossum fibrinogen did not clump a standard preparation of killed staphylococci (Newman D~C coagulase negative) used in the staphylococcal clumping test (Lewis & Wilson, 1973). Fibrinolytic activity was not found in the opossum euglobulin fraction in the presence of streptokinase in a final concentration of 20 or 200/z/ml. Urokinase (100/z/ml) appeared to have a weak activating effect. Bleeding times done on the underbelly skin were less than 1 rain. DISCUSSION Although many of the values observed in the clotting tests differed greatly from those found with human plasma this probably does not reflect any fundamental difference in the coagulation mechanisms. The long times observed with Simplastin® correlate well with the low values in the assays for II, VII and X and could be explained on the lack of activity with this thromboplastin, but it is difficult to understand the high value in the V assay which also employs Simplastin®. The PTT is faster than human, indicating an effective intrinsic mechanism, a finding supported by the T G T test. Platelets showed poor aggregation with collagen, epinephrine and bovine thrombin. This last observation together with the long thrombin time suggests that bovine and opossum materials interreact poorly. That opossum fibrinogen may differ from that of most mammals (Lewis & Wilson, 1973) is supported by the observation of lack of staphylococcal clumping as well as by the long thrombin time. Electron microscopic studies show many ultrastructural characteristics similar to those found in human platelets (White, 1972). About 10~ of the platelets contained fine curly fibrillar material whose character and function are unknown at this time. Acknowledgements--The author wishes to thank Dr. R. Iammarino for advice and for performing the serum electrophoretic and biochemical tests in the clinical laboratories of Presbyterian University Hospital. The author also wishes to thank Ms. Geraldine Morici for her beautiful electron microscope preparations and Ms. Mary Vogel and Messrs. George Arvay, Willie Cooks, James
Donohoe and John Wilson for performing the coagulation studies. REFERENCES DIDISHEIM P., HATFORI K. & LEWIS J. H. (1959) Hematologic and coagulation studies in various animal species, d. Lab. clin. Med. 53, 866-875. FANTLP. (1961) Comparative study of blood coagulation in vertebrates. Austral J. exp. Biol. Med. Sci. 39, 403-412. FANTL P. & WARD H. A. (1957) Comparison of blood clotting in marsupials and man. Austral. J. exp. Biol. Med. Sci. 35, 209-224. GIACOMETTIL., BERNTZENA. & BLISSM. (1972) Hematologic parameters of the opossum (Didelphis virginiana). Comp. Biochem. PhysioL 43A, 287-292. LEWIS J. H. (1961) Coagulation defects. JAMA 178, 1014-1020. LEWIS J. H. (1971) Hemostasis and hemorrhage. Sci. Clin. 1, 1-66. LEWIs J. H. (1974) Comparative hematology: studies on elephants Elephas maximus. Comp. Biochem. PhysioL 49A, 175-181. LEWIs J. H., PHILLIPSL. L. & HANN C. (1968) Coagulation defects in primitive Australian mammals. Comp. Biochem. Physiol. 25, 1129-1135. LEWISJ. H. & WILSONJ. H. (1973) Variations in abilities of animal fibrinogens to clump staphylococci, Thrombosis Res. 3, 419-424. MArS A. & LoEw F. W. (1968) Hemograms of laboratory-confined opossums (Didelphis virginiana). J.A.V.M.A. 153, 800-802. PARSONSR. S. & GUtLER E. R. (1972) Observations on the blood of the marsupial tiger cat, Dasyurops maculatus (Kerr) (Dasyuridae). Comp. Biochem. Physiol. 43A, 935-939. PARSONSR. S., GUILERE. R. & HEDOLER. W. L. (1971) Comparative studies on the blood of monotremes and marsupials--II. Electrolyte, organic constituents, proteins, gas analysis and enzymes. Comp. Biochem. Physiol. 39B, 209-217. PARSONSR. S., HEDDLER. W. L., FLUXW. G. & GUILER E. R. (1970) Studies on the blood of the Tasmanian devil. Comp. Biochem. Physiol. 32, 345-351. ROTHSTEIN R. & HUNSAKERD. (1972) Baseline hematology and blood chemistry of the South American woolly opossum, Caluromys derbianus. Lab. Anita. Sci. 22, 227-232. TIMMONSE. H. & MARQUESP. A. (1969) Blood chemical and hematological studies in the laboratory-confined, unanesthetized opossum, Didelphis virginiana. Lab. Anim. Care 19, 342-344. WARINGH., MOIR R. J. & TYNDALE-BISCOEC. H. (1966) Comparative physiology of marsupials. In Advances in Comparative Physiology and Biochemistry (Edited by LOWENSTEINO.), Vol. 2, pp. 237-376. Department of Zoology and Comparative Physiology, University of Birmingham. WHrrE J. G. (1972) Ultrastructural defects in congenital disorders of platelet function. Ann. N.Y. Acad. Sci. 201, 205-233. YOUATTW. G., FAY L. D., HOWE D. L. & HARTE H. D. (1961) Hematologic data on some small mammals. Blood 18, 758-763. Key Word lndex---Comparative Hematology; opossum; Didelphis marsupialis; platelets; blood coagulation.
