1336
merely that of tissue-factor-like thromboplastin or other residual activity in the supernatants but that of the Fvm-c added in the second hour. not
Discussion of Fvui-c has previously been claimed by groups of workers using, respectively, blood leucocyteslO or fibroblasts," and splenic macrophages.12 In those studies, however, the procoagulant activity shown in simple factor-vui assays could have been due entirely to tissue-factor thromboplastin, and it has been pointed out that this was very probably the case.6,13 In our tests this problem of the artefactual shortening of Fvm-c-assay clotting-times by leucocyte thromboplastin has been overcome by largely removing the thromboplastin with P.L.C.9 The Fvni-c-inhibiting activity which is found in the serum of some 10% of human haemophiliacs is due to an antibody and is therefore taken to be immunologically specific. 14 Accordingly, the neutralisation of procoagulant material by human anti-vm-c is widely taken as the definitive evidence for the identity of that material as FVIII-C. As we have obtained this result with two separate human Fvm-c-inhibitor sera and demonstrated that our 4-way test is a valid method for the demonstration and recovery of Fvlii-c in culture supernatants, and that the supernatants contained material which specifically neutralised human anti-vm-c, it appears to us that the incubated leucocytes stimulated with P.H.A. do in fact synthesise and release FVIII-c. The possibility that this is some other very closely related substance is of course not completely excluded. Some preliminary work of ours has also suggested that monocytes alone may be capable of synthesising the Fvm-c activity. Our finding that leucocytes from patients with severe haemophilia can also synthesise normal amounts of apparent FVIII-c in culture was quite unexpected. Nevertheless, it seems analogous to the originally unexpected but now well-established observation that the other part of the factor-vm molecule, Fvm-R.A., is present in normal or even increased amounts in haemophilic patients’ plasma." It is of course difficult to reconcile our finding-which has been consistent in all five hæmophilic patients so far tested-with the virtual absence of Fvm-c from their circulating plasmas. One possible explanation, in terms of the Jacob/Monod hypothesis, might be that, in haemophiliacs, the operon on the X chromosome which codes for the production of Fvm-c is repressed in vivo but that in some way the conditions of our in-vitro cultures lead to de-represssion, allowing normal synthesis of Fvm-c. The "inducer" which causes the depression could be some metabolic product which accumulates during culture, or it could be the removal of some substance during culture. It might even be the P.H.A., but some preliminary observations of ours have suggested that production of the Fvm-c activity might also be possible without P.H.A. If it can be confirmed that the material we have detected is in fact FVIII-C itself-which means that, given the right conditions or stimulus, hxmophilic leucocytes can be induced to produce quite normal of FVIII-c-the implications for new amounts approaches to the treatment of haemophilia will be considerable. The in-vitro
two
These studies were supported by a research grant from the Trent Health Authority to T.E.B. We thank Dr P. J. Kingston for providing one of the inhibitor-containing sera and the hsemophilic patients and normal volunteers for the blood-samples.
Regional
Requests for reprints should be addressed to T.E.B., Department of Ha:matology, General Hospital, Nottingham, NG1 6HA. REFERENCES
synthesis
1. 2.
Jaffe,
E.
A., Hoyer, L. W., Nachman, R. L. J. clin. Invest. 1973, 52, 2757.
Bloom, A. L. in Recent Advances in Poller); p. 159. Edinburgh, 1977.
Blood
Coagulation,
no.
2 (edited by L.
3. Webster, W. P., Penick, G. D., Mandel, S. R. in Hæmophilia (edited Ala and K. W. E. Denson); p.33. Amsterdam, 1973. 4. Dodds, W. J. ibid. p. 39. 5. Brachman, P., Snyder, J., Henderson, E. S., Astrup, T. Br. J. Hœmat.
by F. 1970,
18, 135. 6. Rickles, F. R., Hardin, J. A., Pitlick, F. A., Hoyer, L. W., Conrad, M. E. J. clin. Invest. 1973, 52, 1427. 7. Blecher, T. E., Thompson, M. J. J. clin. Path. 1976, 29, 727. 8. Hardisty, R. M., Ingram, G. I. C. Bleeding Disorders, Investigation and Management; p. 304. Oxford, 1965. 9. Blecher, T. E., Thompson, M. J., Westby, J. C. Unpublished. 10. Zacharski, L. R., Bowie, E. J. W., Titus, J. L., Owen, C. A. Proc. Staff Meet. Mayo Clin. 1968, 43, 617. 11. Zacharski, L. R., Bowie, E. J. W., Titus, J. L., Owen, C. A. ibid. 1969, 44, 784. 12. Ponn, R. B.,
Kellogg, E. A., Korff, J. M., Pegg, C. A. S., Sise, H. S., NorJ. C. Archs Surg. 1971,103, 398. 13. Bloom, A. L. in Recent Advances in Blood Coagulation, no. 2 (edited by L. Poller); p. 157. Edinburgh, 1977. 14. Bloom, A. L. ibid. p. 142. 15. Zimmerman, T. S., Ratnoff, O. D., Powell, A. E. J. clin Invest. 1971, 50, man,
244.
CHANGES IN BLOOD COAGULATION DURING TOTAL HIP REPLACEMENT G. R. HOUGHTON
Nuffield Orthopœdic Centre, Headington, Oxford OX3 E. G. PAPADAKIS
Hœmophilia Centre,
7LD
C. R. RIZZA
Churchill Hospital, OX7 6NJ
Headington, Oxford
In seventeen patients with osteoarthrosis of the hip undergoing total hip replacement clotting factors II, V, and VIII were measured in blood taken from the operated limb and the general systemic circulation during surgery. Concentrations of these clotting factors were consistently significantly higher in the operated limb than in the rest of the circulation. This result indicates that measures to reduce venostasis in the operated leg during surgery may help to prevent deep-vein thrombosis,
Summary
Introduction DEEP-VEIN thrombosis (D.v.T.) and pulmonary embolus continue to be common causes of morbidity and mortality after total hip replacement. The reported incidence of D.V.T. after hip surgery varies from 34%1 to 83%2. Necropsy studies on patients dying during the postoperative period indicate that pulmonary embolism is the most common cause of death after reconstructive surgery of the hip.3,4 D.V.T. is more common in the operated limb than in the non-operated side. 1,5.6 The factors predisposing patients to venous thrombosis have been known for over a century.’ The triad of vessel-wall damage, venostasis, and changes in blood constituents, probably all contribute to D.v.T. during ,
1337 total hip replacement. Damage to the vessel wall is likely after forceful manipulation, sustained retraction, and prolonged recumbency while supine. Venostasis occurs during dislocation of the hip and is probably exacerbated by retraction to expose the acetabulum. Obstruction of the femoral vein has been demonstrated when the hip is dislocated during total hip replacement.8,9 Limb venostasis causes a pronounced rise in clotting factors v and VIII.I0-12 This effect was confined to the "occluded limb" and did not affect the general circulation. 13 We investigated whether there was a rise in clotting factors II, v, and vin in the operated limb at the period of maximum venostasis during total hip replacement.
HAMATOLOGICAL DATA IN
17
PATIENTS UNDERGOING TOTAL HIP
REPLACEMENT
Patients and Methods
patients (aged 53-73 years, mean 67-5) undergoing hip replacement for osteoarthrosis were studied. Patients had not received any anti-inflammatory drugs for a week before surgery. There were eight men and nine women in the series. No patient had a history of D.V.T., haemorrhagic disorder, or malignant disease. None of the patients had received preoperative or peroperative anticoagulants. All patients had a standard Charnley total hip replacement via the lateral approach and with removal of the greater trochanter .14 The entire leg was prepared. The drapes were placed so that the dorsum of the foot could be exposed during the operation. During cement fixation of the femoral component, the hip is adducted 90 degrees across the opposite thigh. This position Is maintained for approximately 8 min to permit final reaming of the femoral shaft, insertion of the cement and prosthesis, and polymerisation of the methyl methacrylate bone cement. At the end of this period about 15 ml of venous blood was withdrawn simultaneously from a dorsal vein in the foot and from the ipsilateral arm, 9 ml of each sample was mixed with Seventeen
.
3-8% trisodium citrate and the 5 ml
was
mixed with edetic acid
anticoagulant. Five samples with edetic acid were not analysed because of clot formation. Consent was obtained from all patients before surgery. The contralateral foot was rejected as a site for control blood-samples because this would have meant difficult manipulations within the Charnley enclosure and the additional skin exposure might have added to the risk of infecas
tion. The
following were determined in samples from the arm and leg: hxmoglobin, hsematocrit, platelet-count, and factors n, v, and VIII. All were measured within 2 h of collecting the bloodsamples. The haemoglobin, hsematocrit, and platelet-count were estimated with a Coulter counter. Factor II was assayed by the Taipan method," factor v by a one-stage assay based on the one-stage prothrombin-time,16 and factor viii by a twostage method.17 For each estimation the arm sample was taken as
the standard.
Results
haemoglobin concentration and leg was not significant (P=0.17) (see accompanying table). Similarly, although there was a slight increase in haematocrit in the leg samples, this again was not significant (p=O.09). Platelet-counts were similar in the arm and leg samples. Concentrations of clotting factor in samples from the leg were expressed as a percentage of those from the arm. Concentrations of factors II, v and vin in samples from the leg were significantly higher than in those from the arm (factor II, p
merely that of tissue-factor-like thromboplastin or other residual activity in the supernatants but that of the Fvm-c added in the second hour. not
Discussion of Fvui-c has previously been claimed by groups of workers using, respectively, blood leucocyteslO or fibroblasts," and splenic macrophages.12 In those studies, however, the procoagulant activity shown in simple factor-vui assays could have been due entirely to tissue-factor thromboplastin, and it has been pointed out that this was very probably the case.6,13 In our tests this problem of the artefactual shortening of Fvm-c-assay clotting-times by leucocyte thromboplastin has been overcome by largely removing the thromboplastin with P.L.C.9 The Fvni-c-inhibiting activity which is found in the serum of some 10% of human haemophiliacs is due to an antibody and is therefore taken to be immunologically specific. 14 Accordingly, the neutralisation of procoagulant material by human anti-vm-c is widely taken as the definitive evidence for the identity of that material as FVIII-C. As we have obtained this result with two separate human Fvm-c-inhibitor sera and demonstrated that our 4-way test is a valid method for the demonstration and recovery of Fvlii-c in culture supernatants, and that the supernatants contained material which specifically neutralised human anti-vm-c, it appears to us that the incubated leucocytes stimulated with P.H.A. do in fact synthesise and release FVIII-c. The possibility that this is some other very closely related substance is of course not completely excluded. Some preliminary work of ours has also suggested that monocytes alone may be capable of synthesising the Fvm-c activity. Our finding that leucocytes from patients with severe haemophilia can also synthesise normal amounts of apparent FVIII-c in culture was quite unexpected. Nevertheless, it seems analogous to the originally unexpected but now well-established observation that the other part of the factor-vm molecule, Fvm-R.A., is present in normal or even increased amounts in haemophilic patients’ plasma." It is of course difficult to reconcile our finding-which has been consistent in all five hæmophilic patients so far tested-with the virtual absence of Fvm-c from their circulating plasmas. One possible explanation, in terms of the Jacob/Monod hypothesis, might be that, in haemophiliacs, the operon on the X chromosome which codes for the production of Fvm-c is repressed in vivo but that in some way the conditions of our in-vitro cultures lead to de-represssion, allowing normal synthesis of Fvm-c. The "inducer" which causes the depression could be some metabolic product which accumulates during culture, or it could be the removal of some substance during culture. It might even be the P.H.A., but some preliminary observations of ours have suggested that production of the Fvm-c activity might also be possible without P.H.A. If it can be confirmed that the material we have detected is in fact FVIII-C itself-which means that, given the right conditions or stimulus, hxmophilic leucocytes can be induced to produce quite normal of FVIII-c-the implications for new amounts approaches to the treatment of haemophilia will be considerable. The in-vitro
two
These studies were supported by a research grant from the Trent Health Authority to T.E.B. We thank Dr P. J. Kingston for providing one of the inhibitor-containing sera and the hsemophilic patients and normal volunteers for the blood-samples.
Regional
Requests for reprints should be addressed to T.E.B., Department of Ha:matology, General Hospital, Nottingham, NG1 6HA. REFERENCES
synthesis
1. 2.
Jaffe,
E.
A., Hoyer, L. W., Nachman, R. L. J. clin. Invest. 1973, 52, 2757.
Bloom, A. L. in Recent Advances in Poller); p. 159. Edinburgh, 1977.
Blood
Coagulation,
no.
2 (edited by L.
3. Webster, W. P., Penick, G. D., Mandel, S. R. in Hæmophilia (edited Ala and K. W. E. Denson); p.33. Amsterdam, 1973. 4. Dodds, W. J. ibid. p. 39. 5. Brachman, P., Snyder, J., Henderson, E. S., Astrup, T. Br. J. Hœmat.
by F. 1970,
18, 135. 6. Rickles, F. R., Hardin, J. A., Pitlick, F. A., Hoyer, L. W., Conrad, M. E. J. clin. Invest. 1973, 52, 1427. 7. Blecher, T. E., Thompson, M. J. J. clin. Path. 1976, 29, 727. 8. Hardisty, R. M., Ingram, G. I. C. Bleeding Disorders, Investigation and Management; p. 304. Oxford, 1965. 9. Blecher, T. E., Thompson, M. J., Westby, J. C. Unpublished. 10. Zacharski, L. R., Bowie, E. J. W., Titus, J. L., Owen, C. A. Proc. Staff Meet. Mayo Clin. 1968, 43, 617. 11. Zacharski, L. R., Bowie, E. J. W., Titus, J. L., Owen, C. A. ibid. 1969, 44, 784. 12. Ponn, R. B.,
Kellogg, E. A., Korff, J. M., Pegg, C. A. S., Sise, H. S., NorJ. C. Archs Surg. 1971,103, 398. 13. Bloom, A. L. in Recent Advances in Blood Coagulation, no. 2 (edited by L. Poller); p. 157. Edinburgh, 1977. 14. Bloom, A. L. ibid. p. 142. 15. Zimmerman, T. S., Ratnoff, O. D., Powell, A. E. J. clin Invest. 1971, 50, man,
244.
CHANGES IN BLOOD COAGULATION DURING TOTAL HIP REPLACEMENT G. R. HOUGHTON
Nuffield Orthopœdic Centre, Headington, Oxford OX3 E. G. PAPADAKIS
Hœmophilia Centre,
7LD
C. R. RIZZA
Churchill Hospital, OX7 6NJ
Headington, Oxford
In seventeen patients with osteoarthrosis of the hip undergoing total hip replacement clotting factors II, V, and VIII were measured in blood taken from the operated limb and the general systemic circulation during surgery. Concentrations of these clotting factors were consistently significantly higher in the operated limb than in the rest of the circulation. This result indicates that measures to reduce venostasis in the operated leg during surgery may help to prevent deep-vein thrombosis,
Summary
Introduction DEEP-VEIN thrombosis (D.v.T.) and pulmonary embolus continue to be common causes of morbidity and mortality after total hip replacement. The reported incidence of D.V.T. after hip surgery varies from 34%1 to 83%2. Necropsy studies on patients dying during the postoperative period indicate that pulmonary embolism is the most common cause of death after reconstructive surgery of the hip.3,4 D.V.T. is more common in the operated limb than in the non-operated side. 1,5.6 The factors predisposing patients to venous thrombosis have been known for over a century.’ The triad of vessel-wall damage, venostasis, and changes in blood constituents, probably all contribute to D.v.T. during ,
1337 total hip replacement. Damage to the vessel wall is likely after forceful manipulation, sustained retraction, and prolonged recumbency while supine. Venostasis occurs during dislocation of the hip and is probably exacerbated by retraction to expose the acetabulum. Obstruction of the femoral vein has been demonstrated when the hip is dislocated during total hip replacement.8,9 Limb venostasis causes a pronounced rise in clotting factors v and VIII.I0-12 This effect was confined to the "occluded limb" and did not affect the general circulation. 13 We investigated whether there was a rise in clotting factors II, v, and vin in the operated limb at the period of maximum venostasis during total hip replacement.
HAMATOLOGICAL DATA IN
17
PATIENTS UNDERGOING TOTAL HIP
REPLACEMENT
Patients and Methods
patients (aged 53-73 years, mean 67-5) undergoing hip replacement for osteoarthrosis were studied. Patients had not received any anti-inflammatory drugs for a week before surgery. There were eight men and nine women in the series. No patient had a history of D.V.T., haemorrhagic disorder, or malignant disease. None of the patients had received preoperative or peroperative anticoagulants. All patients had a standard Charnley total hip replacement via the lateral approach and with removal of the greater trochanter .14 The entire leg was prepared. The drapes were placed so that the dorsum of the foot could be exposed during the operation. During cement fixation of the femoral component, the hip is adducted 90 degrees across the opposite thigh. This position Is maintained for approximately 8 min to permit final reaming of the femoral shaft, insertion of the cement and prosthesis, and polymerisation of the methyl methacrylate bone cement. At the end of this period about 15 ml of venous blood was withdrawn simultaneously from a dorsal vein in the foot and from the ipsilateral arm, 9 ml of each sample was mixed with Seventeen
.
3-8% trisodium citrate and the 5 ml
was
mixed with edetic acid
anticoagulant. Five samples with edetic acid were not analysed because of clot formation. Consent was obtained from all patients before surgery. The contralateral foot was rejected as a site for control blood-samples because this would have meant difficult manipulations within the Charnley enclosure and the additional skin exposure might have added to the risk of infecas
tion. The
following were determined in samples from the arm and leg: hxmoglobin, hsematocrit, platelet-count, and factors n, v, and VIII. All were measured within 2 h of collecting the bloodsamples. The haemoglobin, hsematocrit, and platelet-count were estimated with a Coulter counter. Factor II was assayed by the Taipan method," factor v by a one-stage assay based on the one-stage prothrombin-time,16 and factor viii by a twostage method.17 For each estimation the arm sample was taken as
the standard.
Results
haemoglobin concentration and leg was not significant (P=0.17) (see accompanying table). Similarly, although there was a slight increase in haematocrit in the leg samples, this again was not significant (p=O.09). Platelet-counts were similar in the arm and leg samples. Concentrations of clotting factor in samples from the leg were expressed as a percentage of those from the arm. Concentrations of factors II, v and vin in samples from the leg were significantly higher than in those from the arm (factor II, p
