Camp. Eiochem. Physiol. Vol. 102A, No. 3, pp. 507-512, 1992 Printed in Great Britain
0300-9629/92 SS.00 + 0.00 0 1992 Pergamon Press Ltd
COMPARATIVE HEMATOLOGY: STUDIES GUINEA-PIGS (CA VIA PORCELLUS)
ON
JE.WCA H. LEWIS*
Central Blood Bank, Pittsburgh, PA 15219, U.S.A. and Department of Medicine, University of Pittsburgh, Pittsburgh, PA 15261, U.S.A. Telephone: (412) 456-1900 (Received 28 October 1991) Abstract-l. Guinea-pig blood clots rapidly and the clots retract in glass tubes. The prothrombin time is long and the activated partial thromboplastin time short compared to human. The Russel viper venom time is similar to human. 2. Factors VII and X assay at levels far below and factors V, VIII and XII assay far above human levels. Other coagulation factors (fibrinogen, II, IX, XI, Fletcher and Fitzgerald) assay within or close to the human range. 3. The thromboplastin generation test results for guinea-pigs and humans are similar. 4. Platelets are numerous and small. They aggregate with ADP, arachidonic acid and pig plasma, variably with ristocetin and poorly with bovine collagen or thrombin. On electron microscopy, platelets appear small with many dark granules (dense bodies). There is an open canicular system. Glycogen particles are sparse. Microtubules are occasionally seen, mitochondria are rare and a-granules are not readily distinguished from dark granules. 5. Ristocetin cofactor is very low, assaying at < 16% of human (co.16 U/ml). 6. Leukocyte counts are variable (630&17,000 per ~1) and differential counts show neutrophils slightly lower and lymphocytes slightly higher than average human counts. 7. Guinea-pig erythrocyte parameters fall within human ranges. 8. Protein electrophoresis shows total protein and albumin to be slightly lower than human. 9. Antithrombin III, Protein C and a,-antiplasmin assay within the human range and plasminogen at very low levels. 10. Bleeding times are consistently about 4 min.
INTRODUCTION
The mammalian order Rodentia contains many inbred domesticated species used as laboratory models in biomedical research. These include the guinea-pig, Cavia porcellus, which was derived from the wild species Cavia culteri. First raised in Peru, the
Incas used the guinea-pig for food and as sacrifices to their gods (Hills, 1972). They were exported to Europe in the 1500s and raised as pets for centuries. Dunkin et al. (1930) described the inbred laboratory strain now called “Hartley”. The guinea-pigs used in this study were outbred from that original strain by the Charles River Breeding Laboratory. Previously reported coagulation findings in guineapigs have shown very long prothrombin times (Chenkin et al., 1959; Hwang and Wosilait, 1970; Hassoun et al., 1989) and low levels of factor VII (Chenkin and Weiner, 1965; Hurt and Krigman, 1970). Dodds and Pickering (1972) examined 10 normal guinea-pigs of each sex before an experiment using cobra venom. Their control values for hematocrit (HCT), hemoglobin (Hgb), white blood cell count (WBC), prothrombin time (PT), activated partial thromboplastin time (APTT) and fibrinogen agree with our findings. Their factor value levels are standardized to a guinea-pig mean value for each sex *Correspondence address: Central Blood Bank, 812 Fifth Avenue, Pittsburgh, PA 15219, U.S.A.
and cannot he interpreted as values compared to normal humans. Scarborough (1931), Wintrobe (1933) and King and Lucas (1941) did extensive studies on guinea-pig blood cells. MATERIALS
AMD METHODS
Blood samples were drawn by direct puncture of the heart (auricle) or the descending aorta from 18 (300-500 g) albino Hartley guinea-pigs following sodium nembutal or ketamine anaesthesia. Blood coagulation tests were essentially the same as those described earlier (Lewis, 1974, 1975, 1976) but semi-automated equipment and standard reagents were used. Bleeding time The guinea-pig bleeding times were done by Peter C. Johnson (1991). Department of Plastic and Reconstructive Surgery, University-of Pittsburgh. They were done with a Simulate” foraanon Teknika Co.. Durham. NC. U.S.A.) on the forelimb d&l to a neonatal cuff inflated to 40 mm’Hg. Hair was removed with Naire depilatory cream. A stop watch measured the time from cut until the site was dry. Drops of blood were collected onto Whatman No. 1 filter paper. Coagulation For coagulation studies, the carefully drawn blood was mixed with one-tenth part 0.13 M (3.8%) sodium citrate. in a polystyrene tube. It was then centrifuged at 15OOg (2800 rpm in a T-JdRS centrifuge) for 15min at 4°C. Plasma was transferred to a new polystyrene tube with a 507
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Comparative hematology plastic Beral pipette and promptly tested or frozen at -70°C. The prothrombin and assays for factors II, V, VII and X used Simplastin@ Excel (Organon Teknika Co.). The activated thromboplastin time (AP’IT) and assays of factors VIII, IX, XI, XII, Fletcher and Fitzgerald used Automated APTT (Organon Teknika Co.). Both PT and APTT tests were done on a Coag-a-Mate X2 (Organon Teknika Co., Bio-Medical, Inc.). The substrates were purchased from George King Bio-Medical, Inc. or pheresed from HIV negative prediagnosed patients. Thrombin and reptilase times These tests were done on a 5-channel Coa Screener (American Labor, Durham, NC, U.S.A.) by adding 50~1 reagent to lOOr titrated plasma and recording clotting time. Thrombin, topical (bovine) U.S.P. Thrombinar is used in a stock solution of 1000 U/ml which is diluted to 10 U/ml for the working solution. This is adjusted to give a clotting time of 15 + 0.5 set by testing with normal human plasma. The reptilase time used Atroxin@ (venom from Eothrops atrox) from Sigma Diagnostics, St Louis, MO, U.S.A. The 5 mg vial content is dissolved in 1.6 ml of distilled water. Crotalus horridus horridus venom (CHH) was purchased from the Miami Serpentarium Laboratories, Miami, FL, U.S.A., and dissolved at 5mg/ml of 0.85% sodium chloride. Staphylococcal clumping test Serial dilutions of plasma 1:2 to I:2048 are prepared in 25 ~1 amounts in a microtiter plate and 25 ~1 staphylococcal clumping factor, 10 mg/ml (Sigma Diagnostics), is added to each well. The plate is covered and placed on a rotator for 20min at room temperature, than shaken vigorously and observed for clumping which is graded 0 to 4.
509
Platelet ultrastructure Transmission electron microscopy of glutaraldehyde fixed btiy coat (Lewis, 1975) used a Philips 300 electron microscope. WBC and RBC counts Leukocyte and erythrocyte counts and sizing were done on a Baker Instruments System 9000 from Serono-Baker Diagnostics, Allentown, PA 18103, U.S.A. Miscellaneous tests Tests for antithrombin III, plasminogen and a,-antiplasmin used ILTM tests and the ACLTM 300 plus (Automated Coagulation Laboratory) Systems, Instrumentation Laboratory, Lexington, MA 02173-3190. For antithrombin III, plasma was incubated with a known amount of thrombin in the presence of heparin and the residual thrombin measured by the lysis of a special chromogenic substrate. For plasminogen, the sample was incubated with a known amount of streptokinase and the formed plasmin measured on another chromogenic substrate. For a,-antiplasmin, the sample was incubated with a known concentration of human plasmin and residual plasmin measured on the same substrate. Protein C activity was assayed using a Stachrome Protein C kit and the ACLTM 300 instrument. RESULTS Bleeding times
Bleeding times in 36 normal guinea-pigs done on the arm, leg or paw averaged 3.75 min, none were longer than 5 min. General coagulation studies
Platelet aggregations Platelet aggregations were measured as changes in light transmision of platelet rich plasma (PRP). Exactly 50 ~1 of aggregating agent was added to 450~1 PRP in a cuvette with magnetic stir bar in a Bio-Data Platelet Aggregation Profiler. Time was started and the increase in light transmission was automatically recorded. Aggregating agents. ADP reagent, stock solution 2 x 10-4mol/l and Ristocetin reagent, stock solution 15 mg/ml were from Sigma Diagnostics. Arachidonic acid, stock solution 5 mg/ml and collagen (lyophilized soluble calf skin collagen), stock solution 1.9 mg/ml were from Bio/Data Corporation, Hatboro, PA 19040, U.S.A. Thrombin, thrombin topical (bovine), U.S.P. Thrombinar was from Armour Pharmaceutical Co., Kankakee, IL 60901, U.S.A. Pig plasma was titrated plasma pooled from three pigs and stored frozen at -70°C. It was used at full strength.
Blood samples were taken by cardiac puncture or from the descending aorta of these small mammals. The blood clots rapidly in either glass or silicone tubes. The clots retract except in polystyrene tubes. Clots do not lyse spontaneously. The serum prothrombin times were very long. The PT was long with Simplastinq, a rabbit brain derivative, and somewhat shorter with guinea-pig brain. Russel viper venom times were about the same in guinea-pigs and humans. Remarkably, the APTT tended to be shorter than the PT. Recalcification times were shorter than those of human plasma. The clots were solid and did not lyse alone or in 1% MCA. The thrombin times with either bovine or human thrombin or CHH were longer than human, and reptilase (venom of Bothrops atrox) did not clot guinea-pig plasma.
Table 2. Coagulation
factors
Guinea-pigs Factor 1 OmGfU II V VII $11 W/ml) IX XI XII
Fletcher Fitzgerald XIII (Recip) RCF%
12 200 0.36 5.25 0.07 0.12 4.5 1.0 I .40 6.75 I .40 I .oo 64
0300-9629/92 SS.00 + 0.00 0 1992 Pergamon Press Ltd
COMPARATIVE HEMATOLOGY: STUDIES GUINEA-PIGS (CA VIA PORCELLUS)
ON
JE.WCA H. LEWIS*
Central Blood Bank, Pittsburgh, PA 15219, U.S.A. and Department of Medicine, University of Pittsburgh, Pittsburgh, PA 15261, U.S.A. Telephone: (412) 456-1900 (Received 28 October 1991) Abstract-l. Guinea-pig blood clots rapidly and the clots retract in glass tubes. The prothrombin time is long and the activated partial thromboplastin time short compared to human. The Russel viper venom time is similar to human. 2. Factors VII and X assay at levels far below and factors V, VIII and XII assay far above human levels. Other coagulation factors (fibrinogen, II, IX, XI, Fletcher and Fitzgerald) assay within or close to the human range. 3. The thromboplastin generation test results for guinea-pigs and humans are similar. 4. Platelets are numerous and small. They aggregate with ADP, arachidonic acid and pig plasma, variably with ristocetin and poorly with bovine collagen or thrombin. On electron microscopy, platelets appear small with many dark granules (dense bodies). There is an open canicular system. Glycogen particles are sparse. Microtubules are occasionally seen, mitochondria are rare and a-granules are not readily distinguished from dark granules. 5. Ristocetin cofactor is very low, assaying at < 16% of human (co.16 U/ml). 6. Leukocyte counts are variable (630&17,000 per ~1) and differential counts show neutrophils slightly lower and lymphocytes slightly higher than average human counts. 7. Guinea-pig erythrocyte parameters fall within human ranges. 8. Protein electrophoresis shows total protein and albumin to be slightly lower than human. 9. Antithrombin III, Protein C and a,-antiplasmin assay within the human range and plasminogen at very low levels. 10. Bleeding times are consistently about 4 min.
INTRODUCTION
The mammalian order Rodentia contains many inbred domesticated species used as laboratory models in biomedical research. These include the guinea-pig, Cavia porcellus, which was derived from the wild species Cavia culteri. First raised in Peru, the
Incas used the guinea-pig for food and as sacrifices to their gods (Hills, 1972). They were exported to Europe in the 1500s and raised as pets for centuries. Dunkin et al. (1930) described the inbred laboratory strain now called “Hartley”. The guinea-pigs used in this study were outbred from that original strain by the Charles River Breeding Laboratory. Previously reported coagulation findings in guineapigs have shown very long prothrombin times (Chenkin et al., 1959; Hwang and Wosilait, 1970; Hassoun et al., 1989) and low levels of factor VII (Chenkin and Weiner, 1965; Hurt and Krigman, 1970). Dodds and Pickering (1972) examined 10 normal guinea-pigs of each sex before an experiment using cobra venom. Their control values for hematocrit (HCT), hemoglobin (Hgb), white blood cell count (WBC), prothrombin time (PT), activated partial thromboplastin time (APTT) and fibrinogen agree with our findings. Their factor value levels are standardized to a guinea-pig mean value for each sex *Correspondence address: Central Blood Bank, 812 Fifth Avenue, Pittsburgh, PA 15219, U.S.A.
and cannot he interpreted as values compared to normal humans. Scarborough (1931), Wintrobe (1933) and King and Lucas (1941) did extensive studies on guinea-pig blood cells. MATERIALS
AMD METHODS
Blood samples were drawn by direct puncture of the heart (auricle) or the descending aorta from 18 (300-500 g) albino Hartley guinea-pigs following sodium nembutal or ketamine anaesthesia. Blood coagulation tests were essentially the same as those described earlier (Lewis, 1974, 1975, 1976) but semi-automated equipment and standard reagents were used. Bleeding time The guinea-pig bleeding times were done by Peter C. Johnson (1991). Department of Plastic and Reconstructive Surgery, University-of Pittsburgh. They were done with a Simulate” foraanon Teknika Co.. Durham. NC. U.S.A.) on the forelimb d&l to a neonatal cuff inflated to 40 mm’Hg. Hair was removed with Naire depilatory cream. A stop watch measured the time from cut until the site was dry. Drops of blood were collected onto Whatman No. 1 filter paper. Coagulation For coagulation studies, the carefully drawn blood was mixed with one-tenth part 0.13 M (3.8%) sodium citrate. in a polystyrene tube. It was then centrifuged at 15OOg (2800 rpm in a T-JdRS centrifuge) for 15min at 4°C. Plasma was transferred to a new polystyrene tube with a 507
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Comparative hematology plastic Beral pipette and promptly tested or frozen at -70°C. The prothrombin and assays for factors II, V, VII and X used Simplastin@ Excel (Organon Teknika Co.). The activated thromboplastin time (AP’IT) and assays of factors VIII, IX, XI, XII, Fletcher and Fitzgerald used Automated APTT (Organon Teknika Co.). Both PT and APTT tests were done on a Coag-a-Mate X2 (Organon Teknika Co., Bio-Medical, Inc.). The substrates were purchased from George King Bio-Medical, Inc. or pheresed from HIV negative prediagnosed patients. Thrombin and reptilase times These tests were done on a 5-channel Coa Screener (American Labor, Durham, NC, U.S.A.) by adding 50~1 reagent to lOOr titrated plasma and recording clotting time. Thrombin, topical (bovine) U.S.P. Thrombinar is used in a stock solution of 1000 U/ml which is diluted to 10 U/ml for the working solution. This is adjusted to give a clotting time of 15 + 0.5 set by testing with normal human plasma. The reptilase time used Atroxin@ (venom from Eothrops atrox) from Sigma Diagnostics, St Louis, MO, U.S.A. The 5 mg vial content is dissolved in 1.6 ml of distilled water. Crotalus horridus horridus venom (CHH) was purchased from the Miami Serpentarium Laboratories, Miami, FL, U.S.A., and dissolved at 5mg/ml of 0.85% sodium chloride. Staphylococcal clumping test Serial dilutions of plasma 1:2 to I:2048 are prepared in 25 ~1 amounts in a microtiter plate and 25 ~1 staphylococcal clumping factor, 10 mg/ml (Sigma Diagnostics), is added to each well. The plate is covered and placed on a rotator for 20min at room temperature, than shaken vigorously and observed for clumping which is graded 0 to 4.
509
Platelet ultrastructure Transmission electron microscopy of glutaraldehyde fixed btiy coat (Lewis, 1975) used a Philips 300 electron microscope. WBC and RBC counts Leukocyte and erythrocyte counts and sizing were done on a Baker Instruments System 9000 from Serono-Baker Diagnostics, Allentown, PA 18103, U.S.A. Miscellaneous tests Tests for antithrombin III, plasminogen and a,-antiplasmin used ILTM tests and the ACLTM 300 plus (Automated Coagulation Laboratory) Systems, Instrumentation Laboratory, Lexington, MA 02173-3190. For antithrombin III, plasma was incubated with a known amount of thrombin in the presence of heparin and the residual thrombin measured by the lysis of a special chromogenic substrate. For plasminogen, the sample was incubated with a known amount of streptokinase and the formed plasmin measured on another chromogenic substrate. For a,-antiplasmin, the sample was incubated with a known concentration of human plasmin and residual plasmin measured on the same substrate. Protein C activity was assayed using a Stachrome Protein C kit and the ACLTM 300 instrument. RESULTS Bleeding times
Bleeding times in 36 normal guinea-pigs done on the arm, leg or paw averaged 3.75 min, none were longer than 5 min. General coagulation studies
Platelet aggregations Platelet aggregations were measured as changes in light transmision of platelet rich plasma (PRP). Exactly 50 ~1 of aggregating agent was added to 450~1 PRP in a cuvette with magnetic stir bar in a Bio-Data Platelet Aggregation Profiler. Time was started and the increase in light transmission was automatically recorded. Aggregating agents. ADP reagent, stock solution 2 x 10-4mol/l and Ristocetin reagent, stock solution 15 mg/ml were from Sigma Diagnostics. Arachidonic acid, stock solution 5 mg/ml and collagen (lyophilized soluble calf skin collagen), stock solution 1.9 mg/ml were from Bio/Data Corporation, Hatboro, PA 19040, U.S.A. Thrombin, thrombin topical (bovine), U.S.P. Thrombinar was from Armour Pharmaceutical Co., Kankakee, IL 60901, U.S.A. Pig plasma was titrated plasma pooled from three pigs and stored frozen at -70°C. It was used at full strength.
Blood samples were taken by cardiac puncture or from the descending aorta of these small mammals. The blood clots rapidly in either glass or silicone tubes. The clots retract except in polystyrene tubes. Clots do not lyse spontaneously. The serum prothrombin times were very long. The PT was long with Simplastinq, a rabbit brain derivative, and somewhat shorter with guinea-pig brain. Russel viper venom times were about the same in guinea-pigs and humans. Remarkably, the APTT tended to be shorter than the PT. Recalcification times were shorter than those of human plasma. The clots were solid and did not lyse alone or in 1% MCA. The thrombin times with either bovine or human thrombin or CHH were longer than human, and reptilase (venom of Bothrops atrox) did not clot guinea-pig plasma.
Table 2. Coagulation
factors
Guinea-pigs Factor 1 OmGfU II V VII $11 W/ml) IX XI XII
Fletcher Fitzgerald XIII (Recip) RCF%
12 200 0.36 5.25 0.07 0.12 4.5 1.0 I .40 6.75 I .40 I .oo 64
