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Stability of canine factor VIII activity and von Willebrand factor antigen concentration in vitro
Research in VeterinaryScience 1991, 51, 313-316
Stability of canine factor VIII activity and von Willebrand factor antigen concentration in vitro P. D. MANSELL, B. W. PARRY, Department o f Veterinary Science, University o f Melbourne, Werribee, Victoria 3030, Australia
The in vitro stability of canine factor VIII activity, von Willebrand factor antigen concentration and the ratio of these two factors was studied. Samples were stored for up to 48 hours, either as plasma or as whole blood, at 4 °, 20 ° and 37°C. Factor VIII activity was generally stable in both plasma and whole blood samples for up to 48 hours at 4 ° or 20°C. The concentration of yon Willebrand factor antigen was more stable in samples stored as plasma than whole blood, and for a shorter time than factor VIII activity. Consequently, the stability of the ratio of these two factors was relatively poor in vitro.
FACTOR VIII (FVIII, antihaemophilic factor) is the component of the intrinsic cascade of blood coagulation which is deficient in individuals with haemophilia A. von Willebrand factor (vWf) is a plasma protein involved in adhesion of platelets to subendothelial collagen or to other platelets and is the deficient factor in von Willebrand's disease. Assay of FVIII and vWf is necessary for the definitive diagnosis of haemophilia A and von Willebrand's disease. In addition, the ratio of FVIII and vWf in plasma may be used to assist the detection of females heterozygous for the haemophilia A gene, because heterozygotes generally have a lower ratio of FVIII to vWf than normal females (Zimmerman et al 1971, Johnstone and Norris 1984). The assays of canine FVIII and vWf are specialised procedures and there is often a delay between collection of blood samples and their processing in a suitable laboratory. Factor VIII is considered to be one of the most labile of the coagulation factors in human plasma (Hondow et al 1982a). Human vWf is more stable in vitro than is human FVII (Eyster et al 1976, Nilsson et al 1983). In contrast, canine FVIII activity is relatively stable in vitro, both in samples stored as plasma and those stored as whole blood. Samples of canine whole blood stored at 4°C for 48 hours retained 81 per cent of the original FVIII activity (Mansell and Parry 1989). Samples stored at 20°C for 48 hours retained 97 per cent of the original FVIII activity. Benson et al (1983) found that the vWf
antigen (vWf:Ag) concentration was stable for up to nine days in plasma samples stored at 2°C, but only for shorter periods at higher temperatures. Dodds (1978) recommended that canine l~lood samples for coagulation assays should be submitted as fresh frozen plasma maintained at - 20°C or lower. Studies of the stability of canine vWf in whole blood in vitro have not been reported. Similarly, there have been no reports of the stability of the ratio of FVIII and vWf activities in canine blood samples in vitro. The purpose of the present study was to investigate the stability of FVIII activity, vWf:Ag concentration and the ratio of FVIII activity and vWf:Ag concentration (FVIII/vWf ratio) in canine blood samples in vitro. Materials and methods
Twelve adult, clinically normal, mixed breed dogs (six male, six female) were used. Blood samples were collected from a jugular vein, using a plastic syringe containing 1/10 volume of 3"8 per cent sodium citrate. Samples to be stored as plasma were centrifuged at 2000 g for 10 minutes and stored in plastic tubes. Samples to be held as whole blood were stored in plastic tubes and centrifuged just before assay. The samples of plasma and whole blood were held at 4, 20 and 37°C. The degree of haemolysis in stored whole blood samples was assessed visually and graded as nil, slight, moderate or severe. Baseline FVIII activities and vWf:Ag concentrations were determined for each dog within 30 minutes of sample collection. Two samples were assayed, one of which was used as the baseline value for the plasma samples and the other used for the baseline value of the whole blood samples. The FVIII activity and vWf'Ag concentration of stored samples were measured 24 and 48 hours after sample collection. Factor VIII activity was measured by a modified onestage activated partial thromboplastin time as previously described (Parry et al 1988). The concentration of vWf:Ag was measured by immunoelectrophoresis using anti-canine vWf antibodies,
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P. D. Mansell, B. IV. Parry TABLE 1 : Effect of storage temperature and storage time on canine factor VIII activity Storage temperature Plasma 4°C 20°C 37°C Whole blood 4°C 20°C 37°C
0
Time after sample collection (h) 24
48
9 5 . 4 4- 3 . 9 a 95.4 ± 3.9 a 95,4 ± 3.9 a
93.2 ± 5.8 a 8 8 . 5 4- 3 . 4 a 60.9 ± 6.4 b
76.8 ± 2.7 b 74.5 ± 4.2 b 1 6 . 4 4- 6 . 8 c
101 . 7 ± 5 . 1 a 101 . 7 4- 5 . 1 a 101 . 7 ± 5 . 1 a
87-1 ± 3.8 b 93-9 ± 2.5 a 86-1 ± 4.7 a
70.1 ± 4.0 c 89-5 ± 5.2 a 101-2-~ 8.5 a
Values are canine units d l - 1 mean ± SEM, n = 10 Values in the same row which have different superscripts are significantly different (P
raised in rabbits, also as described previously (Mansell and Parry 1991). In addition to the FVIII activity and vWf:Ag concentration, the F V I I I / v W f ratio was calculated. For each of the three variables studied (that is FVIII activity, vWf:Ag concentration and F V I I I / v W f ratio) the mean values for 10 of the dogs at each time (baseline, 24 and 48 hours) were compared within that treatment using a repeated measures analysis of variance (Dixon 1988) and Fisher's least significant difference (Hintze 1988). The effects of type of sample (plasma or whole blood) and of temperature of storage (4 °, 20 ° or 37°C) and the interaction between type of sample and storage temperature were tested using repeated measures analysis of variance and Fisher's least significant difference. In the other two dogs used, vWf:Ag was not detectable 48 hours after collection in whole blood samples stored at 37°C. As this precluded the calculation of the F V I I I / v W f ratio, the data from these two dogs were excluded from the statistical analysis. Results
Mild haemolysis was observed in four samples
stored at 37°C for 24 hours and in two samples stored at 20°C for 48 hours. After storage at 37°C for 48 hours, five samples showed mild haemolysis and a further five showed moderate haemolysis. The results of the stability of FVIII activity in plasma and in whole blood in vitro are presented in Table 1. Interaction between type of sample and storage temperature was significant (P < 0-01) in all three periods considered (0 to 48, 0 to 24 and 24 to 48 hours). Results of the stability of vWf:Ag concentration in plasma and in whole blood are presented in Table 2. Interaction between type of sample and temperature of storage was significant during the period 0 to 48 hours (P < 0.05), but was not significant during the other two periods (P > 0.05). Generally, stability was greater in plasma samples, and in samples stored at lower temperature. The stability of the ratio of FVIII activity and vWf:Ag concentration, in plasma and in whole blood (Table 3), reflected the changes in both FVIII activity and vWf:Ag concentration described above. Interaction between type of sample and temperature of storage was significant during the periods 0 to 48 (P<0"01) and 0 to 24 hours (P <0"05), but was not
TABLE 2: Effect of storage temperature and storage time on canine von Willebrand factor
antigen concentration Storage temperature
0
Time after sample collection (h) 24
48
Plasma 4°C 20°C 37°C
9 3 . 3 ± 1 1 . 8 ab 93.3 ± 11.8 a 93.3 ± 11.8 a
88.1 ± 9.7 a 89.9 ± 10.0 a 102.9 ± 12.2 a
107.0 ± 12-3 b 121 . 7 ± 9 . 1 b 130.4 ± 12.9 b
Whole blood 4°C 20°C 37°C
8 5 . 4 4- 1 0 . 7 ab 8 5 . 4 4- 1 0 . 7 a 8 5 . 4 4- 1 0 . 7 a
102.7 ± 8.2 a 128.7 ± 9-5 b 124.7 ± 7-3 b
81 . 6 4- 7 ' 5 b 1 1 2 . 7 4- 1 0 . 1 b 1 3 6 - 5 4- 1 0 . 4 b
values are canine units d1-1 , mean + SEM, n = 10 Values in the same row which have different superscripts are significantly different (P
Stability of canine blood factors
315
TABLE 3: Effect of storage temperature and storage time on the ratio o f canine factor VIII activity/yon Willebrand factor antigen concentration Storage temperature
0
Time after sample collection (h) 24
48
Plasma 4°C 20°C 37°C
1 - 1 6 4- 0 . 1 5 a 1.16 ± 0.15 a 1-16 ± 0.15 a
1 - 1 4 4- 0 . 1 1 a 1.08 ± 0"11 a 0.64 ± 0-07 b
0.82 ± 0.12 b 0.64 ± 0.06 b 0.12 ± 0.05 c
Whole blood 4°C 20°C 37°C
1-35 ± 0.19 a 1 - 3 5 4- 0 . 1 9 a 1-35 ± 0.19 a
0.88 ± 0.06 b 0.76 ± 0.06 b 0.71 ± 0.05 b
0.93 ± 0.10 b 0.86 ± 0.11 b 0.78 ± 0.08 b
Values are factor VIII activity (canine units d l - 1) divided by von Willebrand factor antigen concentration (canine units dl - 1), mean ± SEM, n = 10 Values in the same row which have different superscripts are significantly different (P
significant during the period 24 to 48 hours. The FVIII/vWf ratio was stable only in plasma samples stored at either 4 ° or 20°C. A p a r t from the absence of vWf:Ag after storage at 37°C for 48 hours in the two dogs excluded from the statistical analysis of the data the changes of FVIII activity, vWf:Ag concentration and the FVIII/vWf ratio were similar to those of the other 10 dogs.
Discussion The current study of the stability of canine FVIII activity in vitro confirms our previous findings (Mansell and Parry 1989) that canine FVIII activity in plasma or whole blood samples stored at 4 ° or 20°C should not change sufficiently to interfere with the laboratory discrimination between normal and haemophilic dogs. The stability of FVIII activity was similar in samples stored as plasma or as whole blood when held at either 4 ° or 20°C. In contrast, FVIII activity was much more labile in plasma held at 37°C than in whole blood samples held at the same temperature. The reasons for this difference are unknown. The occurrence of mild to moderate haemolysis in some of the whole blood samples held at 37°C may provide at least a partial explanation, although the mechanisms of action are not clear. Changes of the sample plasma pH, ion concentration and enzymic activity may have occurred with erythrolysis, which could have affected FVIII activity. The in vitro stability of vWf varies with the assay method which is used (Hondow et a11982b, Nilsson et al 1983). This suggests that qualitative changes of the vWf molecule occur during storage, a conclusion which is confirmed by the two-dimensional immunoelectrophoresis methods used by Hondow et al (1982b). Thus, when a one-dimensional immunoelectrophoresis method is used to assay vWf (as in the
present study), the results may be influenced by qualitative changes of the vWf molecule, although such changes will not be obvious. The vWf molecule has a multimeric structure (McCarroll et al 1987, Fujimura et al 1989) and, consequently, degradation of the molecule may occur either as a result of separation of the multimers into oligomers or more fundamental degradation of the protomers themselves. Breakdown of large multimers into smaller multimers may expose more antigenic sites for bi~ding with antibodies used in immunoelectrophoresis methods, producing an apparent increase in plasma vWf content (Batlle and LopezFernandez 1989). This probably explains the increase in vWf values observed in most treatments over 48 hours in the present study. Destruction of the subunits themselves would be reflected as a reduction in plasma vWf content by immunoelectrophoresis methods as antigenic sites are disrupted. As well as being free in plasma, vWf is associated with platelets and is within endothelial cells (Wagner 1989). Platelets contain between 15 and 25 per cent of the vWf in human platelet rich plasma (Howard et al 1974, Nachman and Jaffe 1975, Zucker et al 1979) localised mainly in the a-granules (Zucker et al 1979). Canine platelets contain smaller amounts of vWf (Wardrop et al 1987). Parker et al (1991) calculated that only 3 per cent of the total circulating vWf:Ag was contained in canine platelets. The presence of platelets in samples may result in either release of platelet associated vWf into the plasma, or the absorption of free vWf onto the surface of platelets (Lopez-Fernandez et al 1982). Hence, both increased and decreased concentrations of vWf:Ag in samples stored as whole blood may be explained. However, the small proportion of canine vWf:Ag which is within platelets suggests that release of vWf from platelets into the plasma is of relatively minor importance.
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P. D. Manse& B. W. Parry
Although Nilsson et al (1983) suggested that enzymes released from platelets and erythrocytes contributed to proteolytic degradation of coagulation factors in stored whole blood, the haemolysis seen in some of the samples stored as whole blood at 37 °C did not appear to be associated with reduced FVIII activity or vWf:Ag concentration in these samples. The changes of vWf:Ag concentration during sample storage observed in the present study, although often statistically significant, should not cause a normal dog to be misdiagnosed as suffering from von Willebrand's disease. Because changes of the vWf'Ag concentration were most marked in samples stored as whole blood or at higher temperatures, samples should ideally be submitted as cool plasma and arrive at the laboratory within 24 hours of collection. The F V I I I / v W f ratio changed substantially over the first 24 hours of the study as the FVIII activity decreased and the vWf:Ag concentration increased, although changes in the F V I I I / v W f ratio were generally less pronounced in samples stored as plasma and in those stored at 4 ° or 20°C. The effect of these changes on the accuracy of detection of carriers of the haemophilia A gene would depend on the discriminatory methods used (Mansell 1991). As any variation in the F V I I I / v W f ratio due to sample deterioration is likely to increase the overlap of ratio values between normal and carrier bitches, sample storage will probably decrease the accuracy of any such methods, perhaps to such a degree that laboratory methods of discrimination become clinically worthless. Acknowledgement
The technical assistance of Ms Cheryl Evans and the statistical advice of Mr Garry Anderson were much appreciated. References BATLLE, J. & LOPEZ-FERNANDEZ, M. F. (1989) Coagulation and bleeding disorders: The role of Factor VIII and von Willebrand Factor. Eds T. S. Zimmerman and Z. M. Ruggeri. New York, Marcel Dekker. pp 325-342 BENSON, R. E., JONES, D. W. & DODDS, W. J. (1983) Efficiency and precision of electroimmunoassay for canine factor VIIIrelated antigen. American Journal of Veterinary Research 44, 399-403 DIXON, W. J. (1988) BMDP Statistical Software Manual. University of California Press. pp 483-519 DODDS, W. J. (1978) Inherited bleeding disorders. Canine Practice 5, 49-58 EYSTER, M. E., JONES, M. B., MOORE, T. & DELLI-BOVI, L. (1976) Carrier detection in classic hemophilia by combined measurement of immunologic (VIll AGN) and procoagulant (VIII AHF) activities. American Journal of Clinical Pathology 65, 975 -981
FUJIMURA,Y., RUGGERI, Z. M. & ZIMMERMAN, T.S. (1989) Coagulation and bleeding disorders: The role of Factor VIII and yon Willebrand Factor. Eds T. S. Zimmerman and Z. M. Ruggeri. New York, Marcel Dekker. pp 77-97 HINTZE, J. L. (1988) Solo Statistical Systems Version 2.0 Advanced Set. BMDP Statistical Software Inc. pp 51-66 HONDOW, J. A., RUSSELL, W. J., DUNCAN, B. M. & LLOYD, J. V. (1982a) The stability of coagulation factors in stored blood. Australian and New Zealand Journal of Surgery 52, 265-269 HONDOW, J. A., RUSSELL, W. J., TUNBRIDGE, L. J. & LLOYD, J. V. (1982b) Stability of von Willebrand factor in blood stored at 4 degrees C. Thrombosis Research 27, 125-130 HOWARD, M. A., MONTGOMERY, D. C. & HARDISTY, R. M. (1974) Factor VIII related antigen in platelets. Thrombosis Research 4, 617-624 JOHNSTONE, I. B. & NORRIS, A. M. (1984) A moderately severe expression of classical hemophilia in a family of German shepherds. Canadian Veterinary Journal 25, 191-194 LOPEZ-FERNANDEZ, M. J., GINSBERG, M. H., RUGGERI, Z. M., BATLLE, F. J. & ZIMMERMAN, T. S. (1982) Multimeric structure of platelet factor VIII/vW factor: the presence of larger multimers and their reassociation with thrombin-stimulated platelets. Blood 60, 1132-1138 MANSELL, P. D. (1991) Diagnosis of haemophilia A in dogs, with particular reference to the disease in German shepherd dogs in Australia. PhD thesis, University of Melbourne, Australia MANSELL, P. D. & PARRY, B. W. (1989) Stability of canine factor VIII: coagulant in vitro. Canadian Journal of Veterinary Research 53, 264-267 MANSELL, P. D. & PARRY, B. W. (1991) Changes in Factor VIII: coagulant activity and von Willebrand factor antigen concentration after subcutaneous injection of desmopressin in dogs with mild hemophilia A. Journal of Veterinary lnternal Medicine 5, 191-194 McCARROLL, D. R., LOTHROP, S. A., DOLAN, M. C. & McDONALD, T. P. (1987) Canine von Willebrand factor expresses a multimeric composition similar to human von Willebrand factor. Experimental Hematology 15, 1060-1067 NACHMAN, R. L. & JAFFE, E. A. (1975) Subcellular platelet factor VIII antigen and von Willebrand factor. Journal of Experimental Medicine 141, 1101-1113 NILSSON, L., HEDNER, U., NILSSON, I. M. & ROBERTSON, B. (1983) Shelf-life of bank blood and stored plasma with special reference to coagulation factors. Transfusion 23, 377-381 PARKER, M. T., TURRENTINE, M. A. & JOHNSON, G. S. (1991 ) yon Willebrand factor in lysates of washed canine platelets. American Journal of Veterinary Research 52, 119-125 PARRY, B. W., HOWARD, M. A., MANSELL, P. D. & HOLLOWAY, S. A. (1988) Haemophilia A in German shepherd dogs in Australia. Australian Veterinary Journal 65, 276-279 WAGNER, D. D. (1989) Coagulation and bleeding disorders: The role of Factor VIII and von Willebrand Factor. Eds T. S. Zimmerman and Z. M. Ruggeri. New York, Marcel Dekker. pp 161-180 WARDROP, K. J., MEYERS, K. M. & HELNICK, C. M. (1987) Immunofluorescence studies of factor VIII related antigen in canine vascular endothelium, megakaryocytes and platelets. Proceedings of the American Society of Veterinary Clinical Pathologists. p 29 ZIMMERMAN, T. S., RATNOFF, O. D. & LITTELL, A. S. (1971) Detection of carriers of classic hemophilia using an immunological assay for antihemophilic factor (factor VIII). Journal of Clinical Investigation 50, 255-258 ZUCKER, M. B., BROEKMAN, M. J. & KAPLAN, K. L. (1979) Factor Vlll-related antigen in human blood platelets. Journal of Laboratory and Clinical Medicine 94, 675-682
Received April 8, 1991 Accepted July 4, 1991
Stability of canine factor VIII activity and von Willebrand factor antigen concentration in vitro P. D. MANSELL, B. W. PARRY, Department o f Veterinary Science, University o f Melbourne, Werribee, Victoria 3030, Australia
The in vitro stability of canine factor VIII activity, von Willebrand factor antigen concentration and the ratio of these two factors was studied. Samples were stored for up to 48 hours, either as plasma or as whole blood, at 4 °, 20 ° and 37°C. Factor VIII activity was generally stable in both plasma and whole blood samples for up to 48 hours at 4 ° or 20°C. The concentration of yon Willebrand factor antigen was more stable in samples stored as plasma than whole blood, and for a shorter time than factor VIII activity. Consequently, the stability of the ratio of these two factors was relatively poor in vitro.
FACTOR VIII (FVIII, antihaemophilic factor) is the component of the intrinsic cascade of blood coagulation which is deficient in individuals with haemophilia A. von Willebrand factor (vWf) is a plasma protein involved in adhesion of platelets to subendothelial collagen or to other platelets and is the deficient factor in von Willebrand's disease. Assay of FVIII and vWf is necessary for the definitive diagnosis of haemophilia A and von Willebrand's disease. In addition, the ratio of FVIII and vWf in plasma may be used to assist the detection of females heterozygous for the haemophilia A gene, because heterozygotes generally have a lower ratio of FVIII to vWf than normal females (Zimmerman et al 1971, Johnstone and Norris 1984). The assays of canine FVIII and vWf are specialised procedures and there is often a delay between collection of blood samples and their processing in a suitable laboratory. Factor VIII is considered to be one of the most labile of the coagulation factors in human plasma (Hondow et al 1982a). Human vWf is more stable in vitro than is human FVII (Eyster et al 1976, Nilsson et al 1983). In contrast, canine FVIII activity is relatively stable in vitro, both in samples stored as plasma and those stored as whole blood. Samples of canine whole blood stored at 4°C for 48 hours retained 81 per cent of the original FVIII activity (Mansell and Parry 1989). Samples stored at 20°C for 48 hours retained 97 per cent of the original FVIII activity. Benson et al (1983) found that the vWf
antigen (vWf:Ag) concentration was stable for up to nine days in plasma samples stored at 2°C, but only for shorter periods at higher temperatures. Dodds (1978) recommended that canine l~lood samples for coagulation assays should be submitted as fresh frozen plasma maintained at - 20°C or lower. Studies of the stability of canine vWf in whole blood in vitro have not been reported. Similarly, there have been no reports of the stability of the ratio of FVIII and vWf activities in canine blood samples in vitro. The purpose of the present study was to investigate the stability of FVIII activity, vWf:Ag concentration and the ratio of FVIII activity and vWf:Ag concentration (FVIII/vWf ratio) in canine blood samples in vitro. Materials and methods
Twelve adult, clinically normal, mixed breed dogs (six male, six female) were used. Blood samples were collected from a jugular vein, using a plastic syringe containing 1/10 volume of 3"8 per cent sodium citrate. Samples to be stored as plasma were centrifuged at 2000 g for 10 minutes and stored in plastic tubes. Samples to be held as whole blood were stored in plastic tubes and centrifuged just before assay. The samples of plasma and whole blood were held at 4, 20 and 37°C. The degree of haemolysis in stored whole blood samples was assessed visually and graded as nil, slight, moderate or severe. Baseline FVIII activities and vWf:Ag concentrations were determined for each dog within 30 minutes of sample collection. Two samples were assayed, one of which was used as the baseline value for the plasma samples and the other used for the baseline value of the whole blood samples. The FVIII activity and vWf'Ag concentration of stored samples were measured 24 and 48 hours after sample collection. Factor VIII activity was measured by a modified onestage activated partial thromboplastin time as previously described (Parry et al 1988). The concentration of vWf:Ag was measured by immunoelectrophoresis using anti-canine vWf antibodies,
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P. D. Mansell, B. IV. Parry TABLE 1 : Effect of storage temperature and storage time on canine factor VIII activity Storage temperature Plasma 4°C 20°C 37°C Whole blood 4°C 20°C 37°C
0
Time after sample collection (h) 24
48
9 5 . 4 4- 3 . 9 a 95.4 ± 3.9 a 95,4 ± 3.9 a
93.2 ± 5.8 a 8 8 . 5 4- 3 . 4 a 60.9 ± 6.4 b
76.8 ± 2.7 b 74.5 ± 4.2 b 1 6 . 4 4- 6 . 8 c
101 . 7 ± 5 . 1 a 101 . 7 4- 5 . 1 a 101 . 7 ± 5 . 1 a
87-1 ± 3.8 b 93-9 ± 2.5 a 86-1 ± 4.7 a
70.1 ± 4.0 c 89-5 ± 5.2 a 101-2-~ 8.5 a
Values are canine units d l - 1 mean ± SEM, n = 10 Values in the same row which have different superscripts are significantly different (P
raised in rabbits, also as described previously (Mansell and Parry 1991). In addition to the FVIII activity and vWf:Ag concentration, the F V I I I / v W f ratio was calculated. For each of the three variables studied (that is FVIII activity, vWf:Ag concentration and F V I I I / v W f ratio) the mean values for 10 of the dogs at each time (baseline, 24 and 48 hours) were compared within that treatment using a repeated measures analysis of variance (Dixon 1988) and Fisher's least significant difference (Hintze 1988). The effects of type of sample (plasma or whole blood) and of temperature of storage (4 °, 20 ° or 37°C) and the interaction between type of sample and storage temperature were tested using repeated measures analysis of variance and Fisher's least significant difference. In the other two dogs used, vWf:Ag was not detectable 48 hours after collection in whole blood samples stored at 37°C. As this precluded the calculation of the F V I I I / v W f ratio, the data from these two dogs were excluded from the statistical analysis. Results
Mild haemolysis was observed in four samples
stored at 37°C for 24 hours and in two samples stored at 20°C for 48 hours. After storage at 37°C for 48 hours, five samples showed mild haemolysis and a further five showed moderate haemolysis. The results of the stability of FVIII activity in plasma and in whole blood in vitro are presented in Table 1. Interaction between type of sample and storage temperature was significant (P < 0-01) in all three periods considered (0 to 48, 0 to 24 and 24 to 48 hours). Results of the stability of vWf:Ag concentration in plasma and in whole blood are presented in Table 2. Interaction between type of sample and temperature of storage was significant during the period 0 to 48 hours (P < 0.05), but was not significant during the other two periods (P > 0.05). Generally, stability was greater in plasma samples, and in samples stored at lower temperature. The stability of the ratio of FVIII activity and vWf:Ag concentration, in plasma and in whole blood (Table 3), reflected the changes in both FVIII activity and vWf:Ag concentration described above. Interaction between type of sample and temperature of storage was significant during the periods 0 to 48 (P<0"01) and 0 to 24 hours (P <0"05), but was not
TABLE 2: Effect of storage temperature and storage time on canine von Willebrand factor
antigen concentration Storage temperature
0
Time after sample collection (h) 24
48
Plasma 4°C 20°C 37°C
9 3 . 3 ± 1 1 . 8 ab 93.3 ± 11.8 a 93.3 ± 11.8 a
88.1 ± 9.7 a 89.9 ± 10.0 a 102.9 ± 12.2 a
107.0 ± 12-3 b 121 . 7 ± 9 . 1 b 130.4 ± 12.9 b
Whole blood 4°C 20°C 37°C
8 5 . 4 4- 1 0 . 7 ab 8 5 . 4 4- 1 0 . 7 a 8 5 . 4 4- 1 0 . 7 a
102.7 ± 8.2 a 128.7 ± 9-5 b 124.7 ± 7-3 b
81 . 6 4- 7 ' 5 b 1 1 2 . 7 4- 1 0 . 1 b 1 3 6 - 5 4- 1 0 . 4 b
values are canine units d1-1 , mean + SEM, n = 10 Values in the same row which have different superscripts are significantly different (P
Stability of canine blood factors
315
TABLE 3: Effect of storage temperature and storage time on the ratio o f canine factor VIII activity/yon Willebrand factor antigen concentration Storage temperature
0
Time after sample collection (h) 24
48
Plasma 4°C 20°C 37°C
1 - 1 6 4- 0 . 1 5 a 1.16 ± 0.15 a 1-16 ± 0.15 a
1 - 1 4 4- 0 . 1 1 a 1.08 ± 0"11 a 0.64 ± 0-07 b
0.82 ± 0.12 b 0.64 ± 0.06 b 0.12 ± 0.05 c
Whole blood 4°C 20°C 37°C
1-35 ± 0.19 a 1 - 3 5 4- 0 . 1 9 a 1-35 ± 0.19 a
0.88 ± 0.06 b 0.76 ± 0.06 b 0.71 ± 0.05 b
0.93 ± 0.10 b 0.86 ± 0.11 b 0.78 ± 0.08 b
Values are factor VIII activity (canine units d l - 1) divided by von Willebrand factor antigen concentration (canine units dl - 1), mean ± SEM, n = 10 Values in the same row which have different superscripts are significantly different (P
significant during the period 24 to 48 hours. The FVIII/vWf ratio was stable only in plasma samples stored at either 4 ° or 20°C. A p a r t from the absence of vWf:Ag after storage at 37°C for 48 hours in the two dogs excluded from the statistical analysis of the data the changes of FVIII activity, vWf:Ag concentration and the FVIII/vWf ratio were similar to those of the other 10 dogs.
Discussion The current study of the stability of canine FVIII activity in vitro confirms our previous findings (Mansell and Parry 1989) that canine FVIII activity in plasma or whole blood samples stored at 4 ° or 20°C should not change sufficiently to interfere with the laboratory discrimination between normal and haemophilic dogs. The stability of FVIII activity was similar in samples stored as plasma or as whole blood when held at either 4 ° or 20°C. In contrast, FVIII activity was much more labile in plasma held at 37°C than in whole blood samples held at the same temperature. The reasons for this difference are unknown. The occurrence of mild to moderate haemolysis in some of the whole blood samples held at 37°C may provide at least a partial explanation, although the mechanisms of action are not clear. Changes of the sample plasma pH, ion concentration and enzymic activity may have occurred with erythrolysis, which could have affected FVIII activity. The in vitro stability of vWf varies with the assay method which is used (Hondow et a11982b, Nilsson et al 1983). This suggests that qualitative changes of the vWf molecule occur during storage, a conclusion which is confirmed by the two-dimensional immunoelectrophoresis methods used by Hondow et al (1982b). Thus, when a one-dimensional immunoelectrophoresis method is used to assay vWf (as in the
present study), the results may be influenced by qualitative changes of the vWf molecule, although such changes will not be obvious. The vWf molecule has a multimeric structure (McCarroll et al 1987, Fujimura et al 1989) and, consequently, degradation of the molecule may occur either as a result of separation of the multimers into oligomers or more fundamental degradation of the protomers themselves. Breakdown of large multimers into smaller multimers may expose more antigenic sites for bi~ding with antibodies used in immunoelectrophoresis methods, producing an apparent increase in plasma vWf content (Batlle and LopezFernandez 1989). This probably explains the increase in vWf values observed in most treatments over 48 hours in the present study. Destruction of the subunits themselves would be reflected as a reduction in plasma vWf content by immunoelectrophoresis methods as antigenic sites are disrupted. As well as being free in plasma, vWf is associated with platelets and is within endothelial cells (Wagner 1989). Platelets contain between 15 and 25 per cent of the vWf in human platelet rich plasma (Howard et al 1974, Nachman and Jaffe 1975, Zucker et al 1979) localised mainly in the a-granules (Zucker et al 1979). Canine platelets contain smaller amounts of vWf (Wardrop et al 1987). Parker et al (1991) calculated that only 3 per cent of the total circulating vWf:Ag was contained in canine platelets. The presence of platelets in samples may result in either release of platelet associated vWf into the plasma, or the absorption of free vWf onto the surface of platelets (Lopez-Fernandez et al 1982). Hence, both increased and decreased concentrations of vWf:Ag in samples stored as whole blood may be explained. However, the small proportion of canine vWf:Ag which is within platelets suggests that release of vWf from platelets into the plasma is of relatively minor importance.
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Although Nilsson et al (1983) suggested that enzymes released from platelets and erythrocytes contributed to proteolytic degradation of coagulation factors in stored whole blood, the haemolysis seen in some of the samples stored as whole blood at 37 °C did not appear to be associated with reduced FVIII activity or vWf:Ag concentration in these samples. The changes of vWf:Ag concentration during sample storage observed in the present study, although often statistically significant, should not cause a normal dog to be misdiagnosed as suffering from von Willebrand's disease. Because changes of the vWf'Ag concentration were most marked in samples stored as whole blood or at higher temperatures, samples should ideally be submitted as cool plasma and arrive at the laboratory within 24 hours of collection. The F V I I I / v W f ratio changed substantially over the first 24 hours of the study as the FVIII activity decreased and the vWf:Ag concentration increased, although changes in the F V I I I / v W f ratio were generally less pronounced in samples stored as plasma and in those stored at 4 ° or 20°C. The effect of these changes on the accuracy of detection of carriers of the haemophilia A gene would depend on the discriminatory methods used (Mansell 1991). As any variation in the F V I I I / v W f ratio due to sample deterioration is likely to increase the overlap of ratio values between normal and carrier bitches, sample storage will probably decrease the accuracy of any such methods, perhaps to such a degree that laboratory methods of discrimination become clinically worthless. Acknowledgement
The technical assistance of Ms Cheryl Evans and the statistical advice of Mr Garry Anderson were much appreciated. References BATLLE, J. & LOPEZ-FERNANDEZ, M. F. (1989) Coagulation and bleeding disorders: The role of Factor VIII and von Willebrand Factor. Eds T. S. Zimmerman and Z. M. Ruggeri. New York, Marcel Dekker. pp 325-342 BENSON, R. E., JONES, D. W. & DODDS, W. J. (1983) Efficiency and precision of electroimmunoassay for canine factor VIIIrelated antigen. American Journal of Veterinary Research 44, 399-403 DIXON, W. J. (1988) BMDP Statistical Software Manual. University of California Press. pp 483-519 DODDS, W. J. (1978) Inherited bleeding disorders. Canine Practice 5, 49-58 EYSTER, M. E., JONES, M. B., MOORE, T. & DELLI-BOVI, L. (1976) Carrier detection in classic hemophilia by combined measurement of immunologic (VIll AGN) and procoagulant (VIII AHF) activities. American Journal of Clinical Pathology 65, 975 -981
FUJIMURA,Y., RUGGERI, Z. M. & ZIMMERMAN, T.S. (1989) Coagulation and bleeding disorders: The role of Factor VIII and yon Willebrand Factor. Eds T. S. Zimmerman and Z. M. Ruggeri. New York, Marcel Dekker. pp 77-97 HINTZE, J. L. (1988) Solo Statistical Systems Version 2.0 Advanced Set. BMDP Statistical Software Inc. pp 51-66 HONDOW, J. A., RUSSELL, W. J., DUNCAN, B. M. & LLOYD, J. V. (1982a) The stability of coagulation factors in stored blood. Australian and New Zealand Journal of Surgery 52, 265-269 HONDOW, J. A., RUSSELL, W. J., TUNBRIDGE, L. J. & LLOYD, J. V. (1982b) Stability of von Willebrand factor in blood stored at 4 degrees C. Thrombosis Research 27, 125-130 HOWARD, M. A., MONTGOMERY, D. C. & HARDISTY, R. M. (1974) Factor VIII related antigen in platelets. Thrombosis Research 4, 617-624 JOHNSTONE, I. B. & NORRIS, A. M. (1984) A moderately severe expression of classical hemophilia in a family of German shepherds. Canadian Veterinary Journal 25, 191-194 LOPEZ-FERNANDEZ, M. J., GINSBERG, M. H., RUGGERI, Z. M., BATLLE, F. J. & ZIMMERMAN, T. S. (1982) Multimeric structure of platelet factor VIII/vW factor: the presence of larger multimers and their reassociation with thrombin-stimulated platelets. Blood 60, 1132-1138 MANSELL, P. D. (1991) Diagnosis of haemophilia A in dogs, with particular reference to the disease in German shepherd dogs in Australia. PhD thesis, University of Melbourne, Australia MANSELL, P. D. & PARRY, B. W. (1989) Stability of canine factor VIII: coagulant in vitro. Canadian Journal of Veterinary Research 53, 264-267 MANSELL, P. D. & PARRY, B. W. (1991) Changes in Factor VIII: coagulant activity and von Willebrand factor antigen concentration after subcutaneous injection of desmopressin in dogs with mild hemophilia A. Journal of Veterinary lnternal Medicine 5, 191-194 McCARROLL, D. R., LOTHROP, S. A., DOLAN, M. C. & McDONALD, T. P. (1987) Canine von Willebrand factor expresses a multimeric composition similar to human von Willebrand factor. Experimental Hematology 15, 1060-1067 NACHMAN, R. L. & JAFFE, E. A. (1975) Subcellular platelet factor VIII antigen and von Willebrand factor. Journal of Experimental Medicine 141, 1101-1113 NILSSON, L., HEDNER, U., NILSSON, I. M. & ROBERTSON, B. (1983) Shelf-life of bank blood and stored plasma with special reference to coagulation factors. Transfusion 23, 377-381 PARKER, M. T., TURRENTINE, M. A. & JOHNSON, G. S. (1991 ) yon Willebrand factor in lysates of washed canine platelets. American Journal of Veterinary Research 52, 119-125 PARRY, B. W., HOWARD, M. A., MANSELL, P. D. & HOLLOWAY, S. A. (1988) Haemophilia A in German shepherd dogs in Australia. Australian Veterinary Journal 65, 276-279 WAGNER, D. D. (1989) Coagulation and bleeding disorders: The role of Factor VIII and von Willebrand Factor. Eds T. S. Zimmerman and Z. M. Ruggeri. New York, Marcel Dekker. pp 161-180 WARDROP, K. J., MEYERS, K. M. & HELNICK, C. M. (1987) Immunofluorescence studies of factor VIII related antigen in canine vascular endothelium, megakaryocytes and platelets. Proceedings of the American Society of Veterinary Clinical Pathologists. p 29 ZIMMERMAN, T. S., RATNOFF, O. D. & LITTELL, A. S. (1971) Detection of carriers of classic hemophilia using an immunological assay for antihemophilic factor (factor VIII). Journal of Clinical Investigation 50, 255-258 ZUCKER, M. B., BROEKMAN, M. J. & KAPLAN, K. L. (1979) Factor Vlll-related antigen in human blood platelets. Journal of Laboratory and Clinical Medicine 94, 675-682
Received April 8, 1991 Accepted July 4, 1991