742
test) method. By reference
to the ultraviolet trace the position of the IgE-containing components could be determined,. and from this the sedimentation coefficient could be estimated. We used an empirical relation between sedimentation coefficient and molecular weightS to estimate the molecular weight of the IgE-containing components. A similar ultracentrifugal technique was applied to the measurement of 1251-labelled IgE in which the fractions were counted directly in a gamma counter. IgE assay.-The total IgE content of ultracentrifugation fractions and starting sera were determined by the standard RIST procedure with sheep antibody directed against the IgE myeloma protein N.D.6
Results
patients studied, 15 contained complexed IgE-i.e., IgE with a sedimentation coefficient greater than 8S was detectable. An IgE myeloma (125 I-labelled) protein studied at the same time by a similar technique showed a sharp peak in the region of 8S. A typical ultracentrifugal scan and the distribution of IgE in the corresponding fractions is shown in the accompanying figure, on which has been superimposed a trace of the distribution of radioactivity in a radiolabelled (1251) myeloma IgE preparation ultracentrifuged Of the
sera
of the 16
under identical conditions. Of the group of 16 patients investigated, 10 had atopic eczema and all of these had high-molecular-weight IgE in their sera (see accompanying table). None of the 3 sera from patients with atopic eczema and low serumIgE concentrations (patients 7, 8 and 9) appeared to contain any monomeric IgE, all of the detectable immunoglobulin being in the polymeric form in these sera. Where complexed IgE was present as well as monomer-i.e., in 12 out of 13 of the remaining sera examined-it comprised about 50% of the total IgE (range
I ,
20-70%). Discussion
.
By application of definitive ultracentrifuge analysis we found evidence of complexed IgE in the serum not only of patients with atopic eczema, but also of those with hayfever alone. These complexed forms of IgE do not seem to be formed by aggregation of the immunoglobulin on freezing, since 4 fresh sera also contained complexed IgE. This raised the question of whether these findings are directly related to the disease or whether they reflect a more basic immunological defect. This question can be considered from several points of view. A small number of atopic individuals improve when the offending allergen is removed from their diet-e.g., exclusion of cow’s milk from the diet of some patients with atopic eczema. In this small group, the antigen (possibly 3-lactoglobulin) is the offending agent.On this basis, it is possible that we are observing IgE antigen complexes which are subsequently deposited in the skin of eczema patients thereby causing the lesions, as has been demonstrated in the model system of Spector and Heesom.3 This approach might lead ultimately to the identification of the offending allergen in these patients. Another possibility reflects the functions of rheumatoid factor. Both IgG and IgM antiglobulins (rheumatoid factors) seem to be produced in response to polymeric IgG or, more particularly, as a result of IgG complexed with antigen. Moreover, it has already been shown that IgG antiglobulin can be directed against IgE.7 It would seem possible, therefore, that the com-
demonstrated in our studies could be IgE combined with antiglobulin rather than IgE/allergen complex. Further studies are in progress to investigate this possibility, including ultracentrifuge analysis of sera from allergic patients under conditions known to dissociate antigen/antibody complexes. A third possible interpretation of our observations stems from the work of Soothill and Steward8 who have shown that some animals make poor-affinity antibody which leads to the formation of soluble immune complexes. The inference to be drawn from their studies, and our present findings of complexed IgE in the serum of both the eczematous and hayfever patients, is that atopic patients in general could be producers of pooraffinity IgE antibody. The recent findings of Godfrey9 tend to support this idea. However, complexes (22S) consisting of low-affinity rheumatoid factor (IgM antibody) and IgG become dissociated under similar densitygradient sedimentation conditions to those used in the present study, 10 and we are, therefore, presumably studying complexes containing antibody of much higher affinity. We feel that it is unlikely that atopic patients in general are genetically producers of poor-affinity IgE There is antibody, although this remains a possibility. also no strong HLA linkage with atopy."I Our preliminary findings of what appears to be complexed IgE in the serum of atopic patients raises many questions about the possible presence and nature of other components in the complex. Studies in this direction now in progress could throw further light on the aetiology of atopy. We thank Mrs. J. Wilson for assistance with the IgE estimations.
plexed IgE
The financial support of the Asthma Research Council, and the Arthritis and Rheumatism Council is gratefully acknowledged. J. B. is supported by the Medical Research Council.
Requests for reprints should be addressed to J.B. REFERENCES
1. Turner, M. W., Brostoff, J., Soothill, J. F. Unpublished. 2. Mihm, M. C., Soter, N. A., Dvorak, H., Austen, K. F. J. invest. Derm. 1976,
67, 305. Spector, W. G., Heesom, W. J. Path. Bact 1969, 98, 31. 4. Johns, P., Stanworth, D. R. J. Immun. Meth. 1976, 10, 231. 5. Stewart, G. A., Johns, P. ibid. p. 219. 6. McLaughlan, P., Stanworth, D. R. Lancet, 1975, 64. 7. Williams, R. C., Griffiths, R. W., Emmons, J. D., Field, R. C. J. clin. Invest. 1972, 51, 955. 8. Soothill, J. F., Steward, H. W. Clin. exp. Immun. 1971, 9, 193. 9. Godfrey, R. C. Personal communication. 10. Normansell, D. E., Stanworth, D. R. Immunology, 1966, 10, 527. 11. Turner, M. W., Brostoff, J., Wells, R. S., Stokes, C. R., Soothill, J. F. Clin exp. Immun 1977, 27, 43. 3.
FAILURE OF SYNGENEIC BONE-MARROW GRAFT WITHOUT PRECONDITIONING IN POST-HEPATITIS MARROW APLASIA THE ROYAL MARSDEN HOSPITAL BONE-MARROW TRANSPLANTATION TEAM
Royal Marsden Hospital, Sutton, Surrey A
patient with post-hepatitis bone-maraplasia had bone-marrow transfrom an identical twin. The first two transplants plants were only partially and temporarily successful, but after cyclophosphamide treatment bone-marrow function was
Summary
row
restored. WE report the
problems
of bone-marrow
transplan-
743 tation when one of a pair of identical twins had aplasia after infectious hepatitis. The observations help to explain the pathogenesis of this type of hæmopoienc failure.
Case-report The patient was a girl of 15 when she had a mild attack of hepatitis in September, 1975; in August, 1975, she had stayed in a house where there was a patient with infectious hepatitis. In May, 1976, she complained that she had been bruising easily for some time. Examination showed some bruises and petechial haemorrhages, and a blood-count was: Hb 81 g/1, reticulocytes 2-6%, leucocytes 3-SxlOVl, polymorphs 13%, lymphocytes 87%, platelets 17 x 109/1. The bone-marrow was hypocellular: all the haemopoietic series were represented but megakaryocytes were scarce and there was a reduced number of the more mature cells of both the erythrocytic and granulocytic series. There was, however, a normal number of marrow lymphocytes and plasma-cells together with cells of intermediate character. There was no significant biochemical or other finding; in particular the serum proteins were in no way abnormal. HBAg was not detected. The patient had a twin sister who resembled her very closely, and the probability that they were identical twins was strengthened by the following findings: they had the same red cell antigens (O,Rh CcDee, Ns,Kk, Kp(a-b+), P1+, Le(a-b+). Fy(a+b+), Jk(a+b-), Sd8+, M18,Vw-, Bga-, the same leucocyte antigens (HLA A2,A3,B7,B8, and BW6) and the same serum-globulin allotypes Gm(-1-2,3,5-17-21) (Km(-l). They also had a similar taste threshold for phenylthiocarbamate (P.T.C.). Mixed lymphocyte cultures showed no stimulation. The patient was not seriously ill and the blood-count was not deteriorating, but the ultimate prognosis in post-hepatitis aplasia is known to be poor.’ Because the healthy twin had apparently not had hepatitis and had normal marrow function, it
immediate marrow transplant. On June cells (1.2 x 108/kg) were aspirated from the healthy twin and infused into the patient. The number of cells is lower than is customary for allogeneic grafts but should have been ample for syngeneic grafting. Some recovery of the blood-count was noted in the succeeding 4 weeks (see accompanying figure) and the bone-marrow 21 days after the graft was more cellular, with a higher proportion of hoemopoietic cells. However 5 weeks after transplant she had a mild fever with pharyngitis (the cause of which was not found); there was then a fall in the platelet and neutrophil counts and the marrow, though still cellular, contained fewer hmmopoietic cells and rather more lymphocytes and other mononuclear cells. By Sept. 7, 10 weeks after the transplant, the marrow and blood had returned to their pre-transplant states, but it was not clear whether this apparent rejection was due to the infection, to an insufficient number of grafted cells, or possibly to an inhibitor. On the assumption that an inhibitor was responsible, treatment with azathioprine (200 mg daily for 22weeks) was instituted, but the blood-count continued to fall and there was no improvement in marrow function. However, the patients’ serum, which had previously inhibited the growth of her twin’s bone-marrow colonies (by 61% at a serum concentration of 20%), did not have this effect after the course of azathioprine.2 On Oct. 5, she received a second graft of 1-63x10"’ cells from her twin but, as before, there was only a partial and temporary recovery of haemopoiesis. The failure of this larger number of cells and the earlier findings of an inhibitory factor in the serum indicated that the hypoplasia was more likely to be the result of marrow inhibition than of stem-cell insufficiency. This idea was supported by the finding that marrow taken from the patient 7 weeks after the second transplant grew better in agar after treatment with antithymocyte globulin (A.T.G.).2 Treatment with A.T.G. was considered, but the patient was found to be sensitive to horse-serum, and a second trial of azathioprine was made, with a lower dose for a longer period (100mg daily for 4 weeks). This treatment again reduced the serum factor which inhibited the twin’s marrow in vitro, but only by 20%, and it failed to restore marrow function in vivo. Finally, therefore, the decision was taken to do an orthodox Seattle-type graft with cyclophosphamide preconditioning. This was carried out on March 1, 1977, and was followed by a satisfactory recovery of the blood-count which appears to have stabilised within the normal range. The bone-marrow also shows normal haemopoiesis with fewer lymphoid cells.
was
decided
to
do
24, 1976, 6-3x10’’
an
marrow
Discussion
.
UJ
1977
Blood-counts after each of the three transplants. Ordinate:
azathioprine.
cells/:J.1.
Arrow indicates
day of each transplant. Az:
Where bone-marrow aplasia is due to a deficiency of haemopoietic stem-cells it should always be amenable to treatment by a transplant from a normal identical twin. At least 6 cases have been reported where such a syngeneic transplant was successfully accomplished,34 and where the aplasia was due to drugs or to unknown causes (in 1 case marrow failure was associated with paroxysmal nocturnal haemoglobinuria). However, some syngeneic grafts have failed, sometimes because they were undertaken when the patient was already seriously ill, but, in the cases described by Fernbach and Trentin66 and by Pegg et al. 7, normal haemopoiesis was not restored though the patient lived long enough for this to have happened. Of interest also is a case reported by Speck et awl. in which a syngeneic graft failed to take in a patient who had received immunotherapy for leukaemia. The probable reason for these failures and for the first two graft failures in our case was that the bone-marrow was affected by environmental influences rather than by
744
deficiency of stem-cells. Evidence for the existence of haemopoietic inhibitors acting either on the whole marrow or on one differentiating series has been reported by Krantz9 and by others.’0," AscensaoI2 has also shown that marrow cells from a patient with aplasia would grow more readily in vitro after treatment with A.T.G.; and Hoffman et a1.13 showed that lymphocytes from 5 of 7 patients with aplasia inhibited the growth of erythroid colonies from normal marrows. (It might be objected that these effects were due to a form of allogeneic immunity but that is unlikely. It is discounted in our case a
where the existence of an identical twin allowed tests to be done with a syngeneic system.) Indirect evidence of similar inhibitory mechanisms is provided by the beneficial results of immunosuppressive treatment with cyclophosphamide, azathioprine, or A.T.G. in an impressive number of cases. 14-I8 Our patient has apparently a circulating inhibitor in her serum and the fact that A.T.G.. improved marrow function in vitro may indicate an associated cellmediated immune process. The inhibitor was partially susceptible to azathioprine but the successful restoration of haemopoiesis required pre-conditioning with cyclophosphamide. The dose used was that of the Seattle protocol19 for allogeneic transplants but it is possible that alternative regimens of immunosuppression might be equally successful and better tolerated in cases where there is no compatible donor for marrow. In our case there was a clear history of infectious hepatitis and it seems probable therefore that the virus of that disease is capable of initiating an autoimmune reaction directed against haemopoietic cells, by a mechanism analogous to that of autoimmune thyroditis and similar diseases.
presumably
The Royal Marsden Hospital Bone Marrow Transplant Team includes the following who were concerned with this case: Dr N. M. Blackett, Dr H. McD. Clink, Dr I. D. C. Douglas, Dr M. Gordon, Dr P. F. Howard, Dr B. Jameson, Dr H. E. M. Kay, Dr S. D. Lawler, Sister S. J. Lynch, Dr T. J. McElwain, Dr R. L. Powles, Dr H. G. Prentice, and Dr J. Russell. The patient was diagnosed and referred by Dr F. K. Wright, of the Royal Alexandra Hospital, Rhyl, Clwyd, and we also acknowledge the help pf the medical and technical staff of the Regional Blood Transfusion Service at Liverpool for blood-typing and typing data. We are grateful to the Bud Flanagan Fund for support. Finally, it is appropriate to thank the donor, who sustained three assaults on her marrow with admirable composure.
Correspondence to Dr H. E. M. Kay, Royal Marsden Hospital, Fulham Road, London, SW3 6JJ (but there will be few reprints).
REFERENCES 1. Camitta, B. M., Nathan, D. G., Forman, E. N. Blood, 1974, 43, 473. 2. Gordon, M. Unpublished. 3. Pillow, R. P., Epstein, R. B., Buckner, C. D., Giblett, E. R., Thomas, E. D. New Engl. J. Med. 1966, 275, 94. 4. Thomas, E. D., Rudolph, R. H., Fefer, A., Storb, R., Slichter, S., Buckner, C. D. Exp. Hemat. 1971, 21, 16. 5. Fefer, A., Freeman, H., Storb, R., Hill, J., Singer, J., Edwards, A., Thomas, 6. 7.
E. Ann. intern. Med. 1976, 84, 692. Fernbach, D. J., Trentin, J. J. Proc. int. Congr. Hœtmat. 1950, p. 150. Pegg, D. E., Fleming, W. J. D., Compston, N. Postgrad. med. J. 1964, 40, 213.
8.
Speck, B., Buckner, 105, 1574.
C. D., Jeannet, M.,
et
al. Schweiz. med. Wschr. 1975,
9. Krantz, S. B., Kao, V. Proc. nat. Acad. Sci. 1967, 58, 93. 10. Marmont, A. Blood, 1977, 49, 155. 11. Cline, M. J., Opelz, G., Saxon, A., Fahey, J. L., Golde, D. W. New
J. Med. 1976, 295, 1489.
Methods and Devices AN IMPROVED BLOOD-LOSS MONITOR
West of Scotland Health Boards, Department of Clinical Physics and Bio-Engineering, 11 West Graham Street, Glasgow G4 9LF
R. B. LEWIS
Seafield Childrens Hospital, Ayr R. MCMAHON
Ayrshire Group of Hospitals ONE way to estimate blood-loss during surgery is to wash in the swabs and drapes on which blood has collected and to measure the blood-volume lost by photocolorimetry.’ This is more accurate than the surgeon’s or anæsthetist’s estimate of blood-loss and is easier than weighing the swabs or the patient before and after surgery and red-cell tagging.2 Earlier approaches with the photocolorimeter technique made use of domestic washing-machines, and although accurate results were possible,3 there were disadvantages, including noise and vibration from the machine. Also, circulation of a blood/water mixture through a system of concealed pipes, filters, pumps, and photocolorimeter made proper cleaning impossible. Such a machine was easily choked with debris, making servicing difficult and accuracy doubtful. The new instrument described here overcomes these limitations, making water
blood-loss monitoring continuous, and reliable.
more
convenient, hygienic,
THE INSTRUMENT
The
washing-out system of the blood-loss monitor comprises
tank, an electrically-driven air pump, and an air disperser (see figure). The disperser is a flat perforated vessel set in the
a
tank and connected to the pump by a disposable tube. When the pump is operating, air bubbling through the water creates enough turbulence to wash the blood efficiently from materials placed in the tank. No detergent is needed. Since a small air pump produces enough turbulence, the washing action is quiet and free of vibration. The internal parts of the air disperser are not in contact with the water/blood mixture because air is constantly being blown out. This eliminates clogging by debris. A photocolorimeter probe is immersed in the tank. Intensitymodulated light is split into two beams. One beam passes across the fluid to a light-sensitive detector, and the other passes directly to a reference detector. This system produces accurate and stable readings. The continuous digital display is
Ascensao, J., Kagan, W., Moore, M., Pahwa, R., Hansen, J., Good, R. Lancet, 1976, i, 669. 13. Hoffman, R., Zanjani, E. D., Lutton, J. D., Zalusky, R., Wasserman, L. R. New Engl. J. Med. 1977, 296, 10. 14. Kay, H. E. M., Jameson, B. Proc. R. Soc. Med. 1970, 63, 1075. 15. Baran, D. T., Griner, P. F., Klemperer, M. R. New Engl. J. Med. 1976, 295,
12
1522.
Jeannet, M., Speck, B., Rubinstein, A., Pelet, B., Wyss, M., Kummer, H. Acta hœmat. 1976, 55, 129. 17. Sensenbrenner, L. L., Steele, A. A., Santos, G. W. Exp. Hemat. (in the press). 18. Thomas, E. D., Storb, R., Giblett, E. R., Longpre, B., Weiden, P. L, Fefer, A., Witherspoon, R. A., Buckner, C. D. Exp. Hemat. 1976, 4, 97. 19. Thomas, E. D., Storb, R. Blood, 1970, 36, 507. 16.
Engl.
A. SHAW L. N. GREGORY
J. S. PATON A. D. CUNNINGHAM
test) method. By reference
to the ultraviolet trace the position of the IgE-containing components could be determined,. and from this the sedimentation coefficient could be estimated. We used an empirical relation between sedimentation coefficient and molecular weightS to estimate the molecular weight of the IgE-containing components. A similar ultracentrifugal technique was applied to the measurement of 1251-labelled IgE in which the fractions were counted directly in a gamma counter. IgE assay.-The total IgE content of ultracentrifugation fractions and starting sera were determined by the standard RIST procedure with sheep antibody directed against the IgE myeloma protein N.D.6
Results
patients studied, 15 contained complexed IgE-i.e., IgE with a sedimentation coefficient greater than 8S was detectable. An IgE myeloma (125 I-labelled) protein studied at the same time by a similar technique showed a sharp peak in the region of 8S. A typical ultracentrifugal scan and the distribution of IgE in the corresponding fractions is shown in the accompanying figure, on which has been superimposed a trace of the distribution of radioactivity in a radiolabelled (1251) myeloma IgE preparation ultracentrifuged Of the
sera
of the 16
under identical conditions. Of the group of 16 patients investigated, 10 had atopic eczema and all of these had high-molecular-weight IgE in their sera (see accompanying table). None of the 3 sera from patients with atopic eczema and low serumIgE concentrations (patients 7, 8 and 9) appeared to contain any monomeric IgE, all of the detectable immunoglobulin being in the polymeric form in these sera. Where complexed IgE was present as well as monomer-i.e., in 12 out of 13 of the remaining sera examined-it comprised about 50% of the total IgE (range
I ,
20-70%). Discussion
.
By application of definitive ultracentrifuge analysis we found evidence of complexed IgE in the serum not only of patients with atopic eczema, but also of those with hayfever alone. These complexed forms of IgE do not seem to be formed by aggregation of the immunoglobulin on freezing, since 4 fresh sera also contained complexed IgE. This raised the question of whether these findings are directly related to the disease or whether they reflect a more basic immunological defect. This question can be considered from several points of view. A small number of atopic individuals improve when the offending allergen is removed from their diet-e.g., exclusion of cow’s milk from the diet of some patients with atopic eczema. In this small group, the antigen (possibly 3-lactoglobulin) is the offending agent.On this basis, it is possible that we are observing IgE antigen complexes which are subsequently deposited in the skin of eczema patients thereby causing the lesions, as has been demonstrated in the model system of Spector and Heesom.3 This approach might lead ultimately to the identification of the offending allergen in these patients. Another possibility reflects the functions of rheumatoid factor. Both IgG and IgM antiglobulins (rheumatoid factors) seem to be produced in response to polymeric IgG or, more particularly, as a result of IgG complexed with antigen. Moreover, it has already been shown that IgG antiglobulin can be directed against IgE.7 It would seem possible, therefore, that the com-
demonstrated in our studies could be IgE combined with antiglobulin rather than IgE/allergen complex. Further studies are in progress to investigate this possibility, including ultracentrifuge analysis of sera from allergic patients under conditions known to dissociate antigen/antibody complexes. A third possible interpretation of our observations stems from the work of Soothill and Steward8 who have shown that some animals make poor-affinity antibody which leads to the formation of soluble immune complexes. The inference to be drawn from their studies, and our present findings of complexed IgE in the serum of both the eczematous and hayfever patients, is that atopic patients in general could be producers of pooraffinity IgE antibody. The recent findings of Godfrey9 tend to support this idea. However, complexes (22S) consisting of low-affinity rheumatoid factor (IgM antibody) and IgG become dissociated under similar densitygradient sedimentation conditions to those used in the present study, 10 and we are, therefore, presumably studying complexes containing antibody of much higher affinity. We feel that it is unlikely that atopic patients in general are genetically producers of poor-affinity IgE There is antibody, although this remains a possibility. also no strong HLA linkage with atopy."I Our preliminary findings of what appears to be complexed IgE in the serum of atopic patients raises many questions about the possible presence and nature of other components in the complex. Studies in this direction now in progress could throw further light on the aetiology of atopy. We thank Mrs. J. Wilson for assistance with the IgE estimations.
plexed IgE
The financial support of the Asthma Research Council, and the Arthritis and Rheumatism Council is gratefully acknowledged. J. B. is supported by the Medical Research Council.
Requests for reprints should be addressed to J.B. REFERENCES
1. Turner, M. W., Brostoff, J., Soothill, J. F. Unpublished. 2. Mihm, M. C., Soter, N. A., Dvorak, H., Austen, K. F. J. invest. Derm. 1976,
67, 305. Spector, W. G., Heesom, W. J. Path. Bact 1969, 98, 31. 4. Johns, P., Stanworth, D. R. J. Immun. Meth. 1976, 10, 231. 5. Stewart, G. A., Johns, P. ibid. p. 219. 6. McLaughlan, P., Stanworth, D. R. Lancet, 1975, 64. 7. Williams, R. C., Griffiths, R. W., Emmons, J. D., Field, R. C. J. clin. Invest. 1972, 51, 955. 8. Soothill, J. F., Steward, H. W. Clin. exp. Immun. 1971, 9, 193. 9. Godfrey, R. C. Personal communication. 10. Normansell, D. E., Stanworth, D. R. Immunology, 1966, 10, 527. 11. Turner, M. W., Brostoff, J., Wells, R. S., Stokes, C. R., Soothill, J. F. Clin exp. Immun 1977, 27, 43. 3.
FAILURE OF SYNGENEIC BONE-MARROW GRAFT WITHOUT PRECONDITIONING IN POST-HEPATITIS MARROW APLASIA THE ROYAL MARSDEN HOSPITAL BONE-MARROW TRANSPLANTATION TEAM
Royal Marsden Hospital, Sutton, Surrey A
patient with post-hepatitis bone-maraplasia had bone-marrow transfrom an identical twin. The first two transplants plants were only partially and temporarily successful, but after cyclophosphamide treatment bone-marrow function was
Summary
row
restored. WE report the
problems
of bone-marrow
transplan-
743 tation when one of a pair of identical twins had aplasia after infectious hepatitis. The observations help to explain the pathogenesis of this type of hæmopoienc failure.
Case-report The patient was a girl of 15 when she had a mild attack of hepatitis in September, 1975; in August, 1975, she had stayed in a house where there was a patient with infectious hepatitis. In May, 1976, she complained that she had been bruising easily for some time. Examination showed some bruises and petechial haemorrhages, and a blood-count was: Hb 81 g/1, reticulocytes 2-6%, leucocytes 3-SxlOVl, polymorphs 13%, lymphocytes 87%, platelets 17 x 109/1. The bone-marrow was hypocellular: all the haemopoietic series were represented but megakaryocytes were scarce and there was a reduced number of the more mature cells of both the erythrocytic and granulocytic series. There was, however, a normal number of marrow lymphocytes and plasma-cells together with cells of intermediate character. There was no significant biochemical or other finding; in particular the serum proteins were in no way abnormal. HBAg was not detected. The patient had a twin sister who resembled her very closely, and the probability that they were identical twins was strengthened by the following findings: they had the same red cell antigens (O,Rh CcDee, Ns,Kk, Kp(a-b+), P1+, Le(a-b+). Fy(a+b+), Jk(a+b-), Sd8+, M18,Vw-, Bga-, the same leucocyte antigens (HLA A2,A3,B7,B8, and BW6) and the same serum-globulin allotypes Gm(-1-2,3,5-17-21) (Km(-l). They also had a similar taste threshold for phenylthiocarbamate (P.T.C.). Mixed lymphocyte cultures showed no stimulation. The patient was not seriously ill and the blood-count was not deteriorating, but the ultimate prognosis in post-hepatitis aplasia is known to be poor.’ Because the healthy twin had apparently not had hepatitis and had normal marrow function, it
immediate marrow transplant. On June cells (1.2 x 108/kg) were aspirated from the healthy twin and infused into the patient. The number of cells is lower than is customary for allogeneic grafts but should have been ample for syngeneic grafting. Some recovery of the blood-count was noted in the succeeding 4 weeks (see accompanying figure) and the bone-marrow 21 days after the graft was more cellular, with a higher proportion of hoemopoietic cells. However 5 weeks after transplant she had a mild fever with pharyngitis (the cause of which was not found); there was then a fall in the platelet and neutrophil counts and the marrow, though still cellular, contained fewer hmmopoietic cells and rather more lymphocytes and other mononuclear cells. By Sept. 7, 10 weeks after the transplant, the marrow and blood had returned to their pre-transplant states, but it was not clear whether this apparent rejection was due to the infection, to an insufficient number of grafted cells, or possibly to an inhibitor. On the assumption that an inhibitor was responsible, treatment with azathioprine (200 mg daily for 22weeks) was instituted, but the blood-count continued to fall and there was no improvement in marrow function. However, the patients’ serum, which had previously inhibited the growth of her twin’s bone-marrow colonies (by 61% at a serum concentration of 20%), did not have this effect after the course of azathioprine.2 On Oct. 5, she received a second graft of 1-63x10"’ cells from her twin but, as before, there was only a partial and temporary recovery of haemopoiesis. The failure of this larger number of cells and the earlier findings of an inhibitory factor in the serum indicated that the hypoplasia was more likely to be the result of marrow inhibition than of stem-cell insufficiency. This idea was supported by the finding that marrow taken from the patient 7 weeks after the second transplant grew better in agar after treatment with antithymocyte globulin (A.T.G.).2 Treatment with A.T.G. was considered, but the patient was found to be sensitive to horse-serum, and a second trial of azathioprine was made, with a lower dose for a longer period (100mg daily for 4 weeks). This treatment again reduced the serum factor which inhibited the twin’s marrow in vitro, but only by 20%, and it failed to restore marrow function in vivo. Finally, therefore, the decision was taken to do an orthodox Seattle-type graft with cyclophosphamide preconditioning. This was carried out on March 1, 1977, and was followed by a satisfactory recovery of the blood-count which appears to have stabilised within the normal range. The bone-marrow also shows normal haemopoiesis with fewer lymphoid cells.
was
decided
to
do
24, 1976, 6-3x10’’
an
marrow
Discussion
.
UJ
1977
Blood-counts after each of the three transplants. Ordinate:
azathioprine.
cells/:J.1.
Arrow indicates
day of each transplant. Az:
Where bone-marrow aplasia is due to a deficiency of haemopoietic stem-cells it should always be amenable to treatment by a transplant from a normal identical twin. At least 6 cases have been reported where such a syngeneic transplant was successfully accomplished,34 and where the aplasia was due to drugs or to unknown causes (in 1 case marrow failure was associated with paroxysmal nocturnal haemoglobinuria). However, some syngeneic grafts have failed, sometimes because they were undertaken when the patient was already seriously ill, but, in the cases described by Fernbach and Trentin66 and by Pegg et al. 7, normal haemopoiesis was not restored though the patient lived long enough for this to have happened. Of interest also is a case reported by Speck et awl. in which a syngeneic graft failed to take in a patient who had received immunotherapy for leukaemia. The probable reason for these failures and for the first two graft failures in our case was that the bone-marrow was affected by environmental influences rather than by
744
deficiency of stem-cells. Evidence for the existence of haemopoietic inhibitors acting either on the whole marrow or on one differentiating series has been reported by Krantz9 and by others.’0," AscensaoI2 has also shown that marrow cells from a patient with aplasia would grow more readily in vitro after treatment with A.T.G.; and Hoffman et a1.13 showed that lymphocytes from 5 of 7 patients with aplasia inhibited the growth of erythroid colonies from normal marrows. (It might be objected that these effects were due to a form of allogeneic immunity but that is unlikely. It is discounted in our case a
where the existence of an identical twin allowed tests to be done with a syngeneic system.) Indirect evidence of similar inhibitory mechanisms is provided by the beneficial results of immunosuppressive treatment with cyclophosphamide, azathioprine, or A.T.G. in an impressive number of cases. 14-I8 Our patient has apparently a circulating inhibitor in her serum and the fact that A.T.G.. improved marrow function in vitro may indicate an associated cellmediated immune process. The inhibitor was partially susceptible to azathioprine but the successful restoration of haemopoiesis required pre-conditioning with cyclophosphamide. The dose used was that of the Seattle protocol19 for allogeneic transplants but it is possible that alternative regimens of immunosuppression might be equally successful and better tolerated in cases where there is no compatible donor for marrow. In our case there was a clear history of infectious hepatitis and it seems probable therefore that the virus of that disease is capable of initiating an autoimmune reaction directed against haemopoietic cells, by a mechanism analogous to that of autoimmune thyroditis and similar diseases.
presumably
The Royal Marsden Hospital Bone Marrow Transplant Team includes the following who were concerned with this case: Dr N. M. Blackett, Dr H. McD. Clink, Dr I. D. C. Douglas, Dr M. Gordon, Dr P. F. Howard, Dr B. Jameson, Dr H. E. M. Kay, Dr S. D. Lawler, Sister S. J. Lynch, Dr T. J. McElwain, Dr R. L. Powles, Dr H. G. Prentice, and Dr J. Russell. The patient was diagnosed and referred by Dr F. K. Wright, of the Royal Alexandra Hospital, Rhyl, Clwyd, and we also acknowledge the help pf the medical and technical staff of the Regional Blood Transfusion Service at Liverpool for blood-typing and typing data. We are grateful to the Bud Flanagan Fund for support. Finally, it is appropriate to thank the donor, who sustained three assaults on her marrow with admirable composure.
Correspondence to Dr H. E. M. Kay, Royal Marsden Hospital, Fulham Road, London, SW3 6JJ (but there will be few reprints).
REFERENCES 1. Camitta, B. M., Nathan, D. G., Forman, E. N. Blood, 1974, 43, 473. 2. Gordon, M. Unpublished. 3. Pillow, R. P., Epstein, R. B., Buckner, C. D., Giblett, E. R., Thomas, E. D. New Engl. J. Med. 1966, 275, 94. 4. Thomas, E. D., Rudolph, R. H., Fefer, A., Storb, R., Slichter, S., Buckner, C. D. Exp. Hemat. 1971, 21, 16. 5. Fefer, A., Freeman, H., Storb, R., Hill, J., Singer, J., Edwards, A., Thomas, 6. 7.
E. Ann. intern. Med. 1976, 84, 692. Fernbach, D. J., Trentin, J. J. Proc. int. Congr. Hœtmat. 1950, p. 150. Pegg, D. E., Fleming, W. J. D., Compston, N. Postgrad. med. J. 1964, 40, 213.
8.
Speck, B., Buckner, 105, 1574.
C. D., Jeannet, M.,
et
al. Schweiz. med. Wschr. 1975,
9. Krantz, S. B., Kao, V. Proc. nat. Acad. Sci. 1967, 58, 93. 10. Marmont, A. Blood, 1977, 49, 155. 11. Cline, M. J., Opelz, G., Saxon, A., Fahey, J. L., Golde, D. W. New
J. Med. 1976, 295, 1489.
Methods and Devices AN IMPROVED BLOOD-LOSS MONITOR
West of Scotland Health Boards, Department of Clinical Physics and Bio-Engineering, 11 West Graham Street, Glasgow G4 9LF
R. B. LEWIS
Seafield Childrens Hospital, Ayr R. MCMAHON
Ayrshire Group of Hospitals ONE way to estimate blood-loss during surgery is to wash in the swabs and drapes on which blood has collected and to measure the blood-volume lost by photocolorimetry.’ This is more accurate than the surgeon’s or anæsthetist’s estimate of blood-loss and is easier than weighing the swabs or the patient before and after surgery and red-cell tagging.2 Earlier approaches with the photocolorimeter technique made use of domestic washing-machines, and although accurate results were possible,3 there were disadvantages, including noise and vibration from the machine. Also, circulation of a blood/water mixture through a system of concealed pipes, filters, pumps, and photocolorimeter made proper cleaning impossible. Such a machine was easily choked with debris, making servicing difficult and accuracy doubtful. The new instrument described here overcomes these limitations, making water
blood-loss monitoring continuous, and reliable.
more
convenient, hygienic,
THE INSTRUMENT
The
washing-out system of the blood-loss monitor comprises
tank, an electrically-driven air pump, and an air disperser (see figure). The disperser is a flat perforated vessel set in the
a
tank and connected to the pump by a disposable tube. When the pump is operating, air bubbling through the water creates enough turbulence to wash the blood efficiently from materials placed in the tank. No detergent is needed. Since a small air pump produces enough turbulence, the washing action is quiet and free of vibration. The internal parts of the air disperser are not in contact with the water/blood mixture because air is constantly being blown out. This eliminates clogging by debris. A photocolorimeter probe is immersed in the tank. Intensitymodulated light is split into two beams. One beam passes across the fluid to a light-sensitive detector, and the other passes directly to a reference detector. This system produces accurate and stable readings. The continuous digital display is
Ascensao, J., Kagan, W., Moore, M., Pahwa, R., Hansen, J., Good, R. Lancet, 1976, i, 669. 13. Hoffman, R., Zanjani, E. D., Lutton, J. D., Zalusky, R., Wasserman, L. R. New Engl. J. Med. 1977, 296, 10. 14. Kay, H. E. M., Jameson, B. Proc. R. Soc. Med. 1970, 63, 1075. 15. Baran, D. T., Griner, P. F., Klemperer, M. R. New Engl. J. Med. 1976, 295,
12
1522.
Jeannet, M., Speck, B., Rubinstein, A., Pelet, B., Wyss, M., Kummer, H. Acta hœmat. 1976, 55, 129. 17. Sensenbrenner, L. L., Steele, A. A., Santos, G. W. Exp. Hemat. (in the press). 18. Thomas, E. D., Storb, R., Giblett, E. R., Longpre, B., Weiden, P. L, Fefer, A., Witherspoon, R. A., Buckner, C. D. Exp. Hemat. 1976, 4, 97. 19. Thomas, E. D., Storb, R. Blood, 1970, 36, 507. 16.
Engl.
A. SHAW L. N. GREGORY
J. S. PATON A. D. CUNNINGHAM
