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INCONCLUSIVE IMMUNOHISTOCHEMISTRY OF HUMAN IgE IN MUCOSAL PATHOLOGY
1297 INCONCLUSIVE IMMUNOHISTOCHEMISTRY OF HUMAN IgE IN MUCOSAL PATHOLOGY
SIR,-IgE
is in the focus of’
interest
in relation
to
atopic
or
immediate type-i hypersensitivity reactions.’ Several attempts have been made
to
show local
IgE-forming immunocytes by
immunofluorescence in various diseases.1-!o Some workers an increased number of IgE cells in the lesion to indicate atopic reaction,’5 whereas others base the same conclusion on an apparently reduced number of such cells.’" Notably, the use of more or less unspecified reagents is common to all
take an
these studies.
Lachmann," in 1964, observed the tendency of eosinophilic granulocytes to become non-specifically stained by fluoresceinconjugated proteins. The accompanying table gives some of INFLUENCE OF
CHARACTERISTICS
CONJUGATE CONCENTRATION
AND LABELLING
ON THE NON-SPECIFIC STAINING OF EOSINOPHILIC
GRANULOCYTES IN SECTIONS OF A HUMAN INTESTINAL BIOPSY SPECIMEN
negative (-), negligible (±), distinctly positive (+), and very intensive (++). tF.I.T.c.=f1uorescein isothiocyanate. M.R.I.T.c.=tetramethylrhodamine isothio*Graded:
cvanate.
$These conjugates had been adsorbed with N.D.=not
mouse
liver powder.
determmed.
results with various conjugates applied on paraffin sections12 from the jejunum of a patient with malabsorption and selective IgA deficiency. The degree of labelling is indicated by the optical density (O.D.) ratio of the conjugate-i.e., (o.D. at 280 nm) — (O.D. at Xmax of the bound fluorochrome).13 This ratio can be converted to a molar fluorochrome/protein (F/P) our
ratio only for fluorescein-isothiocyanate-conjugates.13 The table shows the influence of both the protein concentration and the degree of labelling on the staining intensity of eosinophilic granulocytes. From their morphology the fluorescent cells may easily be taken to be specifically stained immunocytes (see figure, a and b). Conjugates of good quality applied at a proper working concentration, however, produce no non-specific staining. This is illustrated in the figure (c) where a distinction between neutrophilic and eosinophilic granulocytes is made by specific (lactoferrin) and noti-specific staining, respectively. Most conjugates used in studies of IgE cells have been subjected to liquid immunoabsorption which introduces another important source of error. All commercial anti-IgE reagents we have tested contained human IgG. This may add to the concentration of highly labelled protein.13 Moreover, such conjugates will contain fluorescent soluble immune complexes which have a high net negative charge and therefore are very prone to produce non-specific staining.12 The complexes may also 1. Lancet, 1975, ii, 1021. 2. Faarup, P., Christensen, E. ibid. 1973, ii, 718. 3. Shiner, M., Ballard, J., Smith, M. E. ibid. 1975, i, 136. 4. Kilby, A., Walker-Smith, J. A., Wood, C. B. S. ibid. 1975, i, 531. 5. Shiner, M., Ballard, J., Brook, C. G. D., Herman, S. ibid. 1975, ii, 1060. 6. Heatley, R. V., Rhodes, J., Calcraft, B. J., Whitehead, R. H., Fifield, R., Newcomb, R. G. ibid. 1975, ii, 1010. 7. Ewen, S. W. B., Munro, A. ibid. 1976, i, 43. 8. Brown, W. R., Borthistle, B. K., Chen, S. T. Clin. exp. Immun. 1975, 20, 227. 9. Green, F. H. Y., Fox, H. Gut, 1975, 16, 125. 10. Lloyd, G., Green, F. H. Y., Fox, H., Mani, V., Turnberg, L. A. ibid. 1975,
16, 861. 11. Lachmann, P. J. in Recent Advances in Clinical Dyke); p. 377. London, 1964. 12. Brandtzaeg, P. Scand. J. Immun. 1973, 2, 333. 13. Brandtzaeg, P. ibid. 1973, 2, 273.
Pathology (edited by
S. C.
Non-specific staining of eosinophilic granulocytes produced by F.I.T.C. and M.R.I.T.C. conjugates in sections of small-intestinal mucosa from patient with malabsorption.
Left column.-Narrow-band excitation
and selective filtration of fluorescence. Right column.-Narrow-band excitation and selective filtration of M.R.I.T.C. fluorescence. (a) Neighbouring sections incubated respectively with human IgG labelled with F.I.T.C. (o.D. ratio 1.5; 1.2 mg/ml; exposure-time 10s) and with M.R.I.T.C. (o.D. ratio 1.8; 0.9 mg/ml; exposure-time 15s). The F.I.T.C. conjugate selectively produced bright non-specific staining of eosinophilic granulocytes, whereas M.R.I.T.C. conjugate also produced faint background staining of epithelium (ep) and lamina proF.I.T.C.
pria. (b) Fields from same two sections to show morphological appearance of eosinophilic granulocytes. Exposure-times lOs. (c) Single section incubated with combination of two rabbit IgG conjugates active, respectively, against human lactoferrin (F.I.T.c.-labelled ; o.D. ratio 3-0; 1.5 mg/ml; exposure-time 10s) and human secretory component (M.R.I.T.C.-labelled; o.D. ratio 5-7; 0.9 mg/ml; exposure-time 15s). Left: four neutrophilic granulocytes showed bright specific staining for lactoferrin, whereas only slight autofluorescence was shown by eosinophilic granulocytes (arrows). Right: same eosinophilic granulocytes showed faint non-specific staining with M.R.I.T.C.
conjugate. (Reduced to ;of x 330 [a]
and 800
[b and ].)
1298 become bound to cells with Fc receptors (for example monocytes) in the tissue, especially when cryostat sections are used. Finally, intestinal immunocytes producing food antibodies may bind fluorescent proteins and thus give false-positive tests for IgE cells. This pitfall is most likely when the conjugate source is goat, sheep, or gumea pig, since heterophil antibodies to gammaglobulins from these species are produced in
man. 14
importance of immunofluorescence performance testing positive and negative substratesf2 15 16 has been neglected in most immunohistochemical studies of IgE. In addition, absorption controls of the conjugate with antigen added in excess should be performed to obtain a good indication about the proneness to unwanted staining since neither immunological specificity nor binding specificity can be fully ensured by The
on
blocking controls with unlabelled antiserum.12 Our rabbit anti-IgE conjugate 15 reveals very
few IgE im18 munocytes in normal and diseased intestinal mucosa. 17 Moreover, there is no staining of the epithelium or of the extracellular areas in the lamina propria. Even with a 1000-fold increase of the IgE level its concentration in the interstitial fluid should be only in the range of 1-20 mg/dl. Experience with other proteins indicates that this concentration would be below the level of detection by immunofluorescence. Moreover, there is no evidence for an active epithelial of IgE.19 Description of extracellular 25 uptake and transport 6 and epithelial3 8 IgE fluorescence therefore hints at poor conjugate quality or at a too high working concentration." Such experiments cannot be trusted in studies of IgE immunocytes. In one report the "IgE-positive" cells were shown to contain peroxidasewhich strongly indicates that the fluorescence represented unwanted staining of granulocytes. We occasionally see a faint peripheral staining of mast cells but have not paid any attention to this observation since these cells exhibit affinity for conjugated rabbit IgG.1O However, FeltkampVroom et al. have claimed that it is possible to demonstrate immunohistochemically IgE on the membrane of mast cells; the staining intensity was related to the clinical diagnosis of atopy rather than to the serum level of IgE .21 We do not dispute that locally produced IgE may be involved in the pathogenesis of some mucosal lesions. However, we urge that further studies in this area should be standardised and based on properly defined and characterised reagents. As discussed in detail elsewhere ’22 conflicting and confusing data abound in relation to mucosal immunopathology; a great step forward would be the elimination of methodological problems. Department of Microbiology, Dental Faculty, University of Oslo, and Immunohistochemical Laboratory, Institute of Pathology, University Hospital, Rikshospitalet, Oslo 1, Norway Medical Department A, University Hospital, Rikshospitalet, Oslo
PER BRANDTZAEG
KÅRE BAKLIEN
INSULIN AND LIVER SIZE AND FUNCTION
SIR,-We read with interest the report by Dr Starzl and his colleagues (April 17, p. 821) which shows that the effects produced by complete diversion of portal flow from the liver can be ameliorated by direct administration of intraportal insulin. It is difficult to separate experimentally the liver atrophy produced by reduction in flow after portal diversion from that Fouchard, T., Bennich, H., Johansson, S. G. O., Lundkvist, U. Int. Archs Allergy appl. Immun. 1975, 48, 812. 15. Brandtzaeg, P. Immunology, 1972, 22, 177. 16. Brandtzaeg, P. Ann. N.Y. Acad. Sci. 1975, 254, 35. 17. Baklien, K., Brandtzaeg, P. Clin. exp. Immun. 1975, 22, 197. 18. Baklien, K., Brandtzaeg, P. Scand. J. Gastroent. 1976, (in the press). 19. Nakajima, S., Gillespie, D. N., Gleich, G. J. Clin. exp. Immun. 1975, 21,
caused by diversion of "hepatotrophic factors" to the systemic circulation. In previous reports,’2 Starzl’s group studied dogs in which portal blood from the upper gastrointestinal tract (including pancreas) was selectively diverted to the right liver lobe and blood from the lower gastrointestinal tract was routed to the left liver lobe. The conclusions drawn from these studies could be criticised in that the changes demonstrated might have been related to the relative volumes of flow rather than to the presence of hepatotrophic factors. However, their latest study reinforces the case for the importance of hepatotrophic factors, especially insulin, in maintaining liver size and function. We have done experiments in the dog using the split splanchnic technique of Starzl et al.’ which allows selective portal diversion, and have measured nutrient blood-flow to the liver using a xenon-133washout technique.3We found, as did Starzl’s group, that the liver lobe supplied by non-pancreatic portal blood atrophies. We also found that liver perfusion to the atrophic lobe is normal, and thus we can support Starzl’s conclusions. We would, however, interpret Starzl’s autoradiographic studies more cautiously. Tritiated thymidine is taken up by the liver cell into protein, fats, and R.N.A. as well as D.N.A.’ It would therefore be wrong to infer from autoradiographs of tissue undergoing striking cytoplasmic change that all activity recorded represented nuclear replication. The fraction of D.N.A. undergoing replication is best assessed by measuring the specific D.N.A. activity5 which we have found to be unchanged in regenerating rat liver perfused by non-portal blood after portacaval transposition (unpublished). It would seem that liver atrophy following portal diversion is probably related to the diversion of contents of portal blood rather than flow but that the D.N.A. synthetic response in the liver after partial hepatectomy is not impaired by diversion of these hepatotrophic factors. D. P. LEIBERMAN Department of Surgery, J. GUEST Royal Infirmary, Glasgow G4 0SF L. H. BLUMGART
CORONARY HEART-DISEASE: TWO THOUGHTS FROM ONE COLLEGE
SiR,—The report6 on prevention of coronary heart-disease by a Working Party of the Royal College of Physicians of London and the British Cardiac Society prompted me to go back to the earlier report on the care of the patient with coronary heart-disease by a separate working-party constituted by the same bodies. The earlier report said (p. 42): "a more positive approach to the problems of sudden unexpected death, and the high mortality in the first hours after the attack, is necessary. More attention must be paid to the organisation of emergency medical services, including the provision of immediate coronary care ...". The later report said (p. 11): "It would seem that the potential benefit from improving emergency services would be disappointingly small ... Most deaths from CHD far too suddenly for present forms of medical attention be effective ...". Six months separated publication of the two reports. What is the average reader to make of two statements that are close to contradicting one another but that come from an identical-and eminent-pair of bodies? I certainly am conoccur
to
14.
306. 20. 21. 22.
Young, S., Cowan, D. M. Br. J. exp. Path. 1965, 46, 649. Feltkamp-Vroom, T. M., Stallman, P. J., Aalberse, R. C., Reerink-Brongers, E. E. Clin. Immun. Immunopath. 1975, 4, 392. Brandtzaeg, P., Baklien, K. Scand. J. Gastroent. 1976, 11, suppl. 36.
1.
Starzl, T. E., Francavilla, A., Halgrimson, C. G., Francavilla, F. R., Porter, K. A., Brown, T. H., Putnam, C. W. Surgery Gynec. Obstet. 1973, 137, 179.
2.
Starzl, T. E., Porter, K. A., Kashiwagi, N. and others. ibid. 1975, 140, 549. 3. Mackenzie, R. J., Leiberman, D. P., Mathie, R. T., Rice, G. C., Harper, A. M., Blumgart, L. H. Acta chir. scand. (in the press). 4. Schneider, W. C., Greco, A. Biochim. biophys. Acta, 1971, 228, 610. 5. Bucher, N. L. R., Swaffield, M. N. Cancer Res. 1973, 33, 3189. 6. Report of a Joint Working Party of the Royal College of Physicians of London and the British Cardiac Society Jl R. Coll. Physns. 1976, 10, 213. 7. ibid. 1975, 10, 5.
SIR,-IgE
is in the focus of’
interest
in relation
to
atopic
or
immediate type-i hypersensitivity reactions.’ Several attempts have been made
to
show local
IgE-forming immunocytes by
immunofluorescence in various diseases.1-!o Some workers an increased number of IgE cells in the lesion to indicate atopic reaction,’5 whereas others base the same conclusion on an apparently reduced number of such cells.’" Notably, the use of more or less unspecified reagents is common to all
take an
these studies.
Lachmann," in 1964, observed the tendency of eosinophilic granulocytes to become non-specifically stained by fluoresceinconjugated proteins. The accompanying table gives some of INFLUENCE OF
CHARACTERISTICS
CONJUGATE CONCENTRATION
AND LABELLING
ON THE NON-SPECIFIC STAINING OF EOSINOPHILIC
GRANULOCYTES IN SECTIONS OF A HUMAN INTESTINAL BIOPSY SPECIMEN
negative (-), negligible (±), distinctly positive (+), and very intensive (++). tF.I.T.c.=f1uorescein isothiocyanate. M.R.I.T.c.=tetramethylrhodamine isothio*Graded:
cvanate.
$These conjugates had been adsorbed with N.D.=not
mouse
liver powder.
determmed.
results with various conjugates applied on paraffin sections12 from the jejunum of a patient with malabsorption and selective IgA deficiency. The degree of labelling is indicated by the optical density (O.D.) ratio of the conjugate-i.e., (o.D. at 280 nm) — (O.D. at Xmax of the bound fluorochrome).13 This ratio can be converted to a molar fluorochrome/protein (F/P) our
ratio only for fluorescein-isothiocyanate-conjugates.13 The table shows the influence of both the protein concentration and the degree of labelling on the staining intensity of eosinophilic granulocytes. From their morphology the fluorescent cells may easily be taken to be specifically stained immunocytes (see figure, a and b). Conjugates of good quality applied at a proper working concentration, however, produce no non-specific staining. This is illustrated in the figure (c) where a distinction between neutrophilic and eosinophilic granulocytes is made by specific (lactoferrin) and noti-specific staining, respectively. Most conjugates used in studies of IgE cells have been subjected to liquid immunoabsorption which introduces another important source of error. All commercial anti-IgE reagents we have tested contained human IgG. This may add to the concentration of highly labelled protein.13 Moreover, such conjugates will contain fluorescent soluble immune complexes which have a high net negative charge and therefore are very prone to produce non-specific staining.12 The complexes may also 1. Lancet, 1975, ii, 1021. 2. Faarup, P., Christensen, E. ibid. 1973, ii, 718. 3. Shiner, M., Ballard, J., Smith, M. E. ibid. 1975, i, 136. 4. Kilby, A., Walker-Smith, J. A., Wood, C. B. S. ibid. 1975, i, 531. 5. Shiner, M., Ballard, J., Brook, C. G. D., Herman, S. ibid. 1975, ii, 1060. 6. Heatley, R. V., Rhodes, J., Calcraft, B. J., Whitehead, R. H., Fifield, R., Newcomb, R. G. ibid. 1975, ii, 1010. 7. Ewen, S. W. B., Munro, A. ibid. 1976, i, 43. 8. Brown, W. R., Borthistle, B. K., Chen, S. T. Clin. exp. Immun. 1975, 20, 227. 9. Green, F. H. Y., Fox, H. Gut, 1975, 16, 125. 10. Lloyd, G., Green, F. H. Y., Fox, H., Mani, V., Turnberg, L. A. ibid. 1975,
16, 861. 11. Lachmann, P. J. in Recent Advances in Clinical Dyke); p. 377. London, 1964. 12. Brandtzaeg, P. Scand. J. Immun. 1973, 2, 333. 13. Brandtzaeg, P. ibid. 1973, 2, 273.
Pathology (edited by
S. C.
Non-specific staining of eosinophilic granulocytes produced by F.I.T.C. and M.R.I.T.C. conjugates in sections of small-intestinal mucosa from patient with malabsorption.
Left column.-Narrow-band excitation
and selective filtration of fluorescence. Right column.-Narrow-band excitation and selective filtration of M.R.I.T.C. fluorescence. (a) Neighbouring sections incubated respectively with human IgG labelled with F.I.T.C. (o.D. ratio 1.5; 1.2 mg/ml; exposure-time 10s) and with M.R.I.T.C. (o.D. ratio 1.8; 0.9 mg/ml; exposure-time 15s). The F.I.T.C. conjugate selectively produced bright non-specific staining of eosinophilic granulocytes, whereas M.R.I.T.C. conjugate also produced faint background staining of epithelium (ep) and lamina proF.I.T.C.
pria. (b) Fields from same two sections to show morphological appearance of eosinophilic granulocytes. Exposure-times lOs. (c) Single section incubated with combination of two rabbit IgG conjugates active, respectively, against human lactoferrin (F.I.T.c.-labelled ; o.D. ratio 3-0; 1.5 mg/ml; exposure-time 10s) and human secretory component (M.R.I.T.C.-labelled; o.D. ratio 5-7; 0.9 mg/ml; exposure-time 15s). Left: four neutrophilic granulocytes showed bright specific staining for lactoferrin, whereas only slight autofluorescence was shown by eosinophilic granulocytes (arrows). Right: same eosinophilic granulocytes showed faint non-specific staining with M.R.I.T.C.
conjugate. (Reduced to ;of x 330 [a]
and 800
[b and ].)
1298 become bound to cells with Fc receptors (for example monocytes) in the tissue, especially when cryostat sections are used. Finally, intestinal immunocytes producing food antibodies may bind fluorescent proteins and thus give false-positive tests for IgE cells. This pitfall is most likely when the conjugate source is goat, sheep, or gumea pig, since heterophil antibodies to gammaglobulins from these species are produced in
man. 14
importance of immunofluorescence performance testing positive and negative substratesf2 15 16 has been neglected in most immunohistochemical studies of IgE. In addition, absorption controls of the conjugate with antigen added in excess should be performed to obtain a good indication about the proneness to unwanted staining since neither immunological specificity nor binding specificity can be fully ensured by The
on
blocking controls with unlabelled antiserum.12 Our rabbit anti-IgE conjugate 15 reveals very
few IgE im18 munocytes in normal and diseased intestinal mucosa. 17 Moreover, there is no staining of the epithelium or of the extracellular areas in the lamina propria. Even with a 1000-fold increase of the IgE level its concentration in the interstitial fluid should be only in the range of 1-20 mg/dl. Experience with other proteins indicates that this concentration would be below the level of detection by immunofluorescence. Moreover, there is no evidence for an active epithelial of IgE.19 Description of extracellular 25 uptake and transport 6 and epithelial3 8 IgE fluorescence therefore hints at poor conjugate quality or at a too high working concentration." Such experiments cannot be trusted in studies of IgE immunocytes. In one report the "IgE-positive" cells were shown to contain peroxidasewhich strongly indicates that the fluorescence represented unwanted staining of granulocytes. We occasionally see a faint peripheral staining of mast cells but have not paid any attention to this observation since these cells exhibit affinity for conjugated rabbit IgG.1O However, FeltkampVroom et al. have claimed that it is possible to demonstrate immunohistochemically IgE on the membrane of mast cells; the staining intensity was related to the clinical diagnosis of atopy rather than to the serum level of IgE .21 We do not dispute that locally produced IgE may be involved in the pathogenesis of some mucosal lesions. However, we urge that further studies in this area should be standardised and based on properly defined and characterised reagents. As discussed in detail elsewhere ’22 conflicting and confusing data abound in relation to mucosal immunopathology; a great step forward would be the elimination of methodological problems. Department of Microbiology, Dental Faculty, University of Oslo, and Immunohistochemical Laboratory, Institute of Pathology, University Hospital, Rikshospitalet, Oslo 1, Norway Medical Department A, University Hospital, Rikshospitalet, Oslo
PER BRANDTZAEG
KÅRE BAKLIEN
INSULIN AND LIVER SIZE AND FUNCTION
SIR,-We read with interest the report by Dr Starzl and his colleagues (April 17, p. 821) which shows that the effects produced by complete diversion of portal flow from the liver can be ameliorated by direct administration of intraportal insulin. It is difficult to separate experimentally the liver atrophy produced by reduction in flow after portal diversion from that Fouchard, T., Bennich, H., Johansson, S. G. O., Lundkvist, U. Int. Archs Allergy appl. Immun. 1975, 48, 812. 15. Brandtzaeg, P. Immunology, 1972, 22, 177. 16. Brandtzaeg, P. Ann. N.Y. Acad. Sci. 1975, 254, 35. 17. Baklien, K., Brandtzaeg, P. Clin. exp. Immun. 1975, 22, 197. 18. Baklien, K., Brandtzaeg, P. Scand. J. Gastroent. 1976, (in the press). 19. Nakajima, S., Gillespie, D. N., Gleich, G. J. Clin. exp. Immun. 1975, 21,
caused by diversion of "hepatotrophic factors" to the systemic circulation. In previous reports,’2 Starzl’s group studied dogs in which portal blood from the upper gastrointestinal tract (including pancreas) was selectively diverted to the right liver lobe and blood from the lower gastrointestinal tract was routed to the left liver lobe. The conclusions drawn from these studies could be criticised in that the changes demonstrated might have been related to the relative volumes of flow rather than to the presence of hepatotrophic factors. However, their latest study reinforces the case for the importance of hepatotrophic factors, especially insulin, in maintaining liver size and function. We have done experiments in the dog using the split splanchnic technique of Starzl et al.’ which allows selective portal diversion, and have measured nutrient blood-flow to the liver using a xenon-133washout technique.3We found, as did Starzl’s group, that the liver lobe supplied by non-pancreatic portal blood atrophies. We also found that liver perfusion to the atrophic lobe is normal, and thus we can support Starzl’s conclusions. We would, however, interpret Starzl’s autoradiographic studies more cautiously. Tritiated thymidine is taken up by the liver cell into protein, fats, and R.N.A. as well as D.N.A.’ It would therefore be wrong to infer from autoradiographs of tissue undergoing striking cytoplasmic change that all activity recorded represented nuclear replication. The fraction of D.N.A. undergoing replication is best assessed by measuring the specific D.N.A. activity5 which we have found to be unchanged in regenerating rat liver perfused by non-portal blood after portacaval transposition (unpublished). It would seem that liver atrophy following portal diversion is probably related to the diversion of contents of portal blood rather than flow but that the D.N.A. synthetic response in the liver after partial hepatectomy is not impaired by diversion of these hepatotrophic factors. D. P. LEIBERMAN Department of Surgery, J. GUEST Royal Infirmary, Glasgow G4 0SF L. H. BLUMGART
CORONARY HEART-DISEASE: TWO THOUGHTS FROM ONE COLLEGE
SiR,—The report6 on prevention of coronary heart-disease by a Working Party of the Royal College of Physicians of London and the British Cardiac Society prompted me to go back to the earlier report on the care of the patient with coronary heart-disease by a separate working-party constituted by the same bodies. The earlier report said (p. 42): "a more positive approach to the problems of sudden unexpected death, and the high mortality in the first hours after the attack, is necessary. More attention must be paid to the organisation of emergency medical services, including the provision of immediate coronary care ...". The later report said (p. 11): "It would seem that the potential benefit from improving emergency services would be disappointingly small ... Most deaths from CHD far too suddenly for present forms of medical attention be effective ...". Six months separated publication of the two reports. What is the average reader to make of two statements that are close to contradicting one another but that come from an identical-and eminent-pair of bodies? I certainly am conoccur
to
14.
306. 20. 21. 22.
Young, S., Cowan, D. M. Br. J. exp. Path. 1965, 46, 649. Feltkamp-Vroom, T. M., Stallman, P. J., Aalberse, R. C., Reerink-Brongers, E. E. Clin. Immun. Immunopath. 1975, 4, 392. Brandtzaeg, P., Baklien, K. Scand. J. Gastroent. 1976, 11, suppl. 36.
1.
Starzl, T. E., Francavilla, A., Halgrimson, C. G., Francavilla, F. R., Porter, K. A., Brown, T. H., Putnam, C. W. Surgery Gynec. Obstet. 1973, 137, 179.
2.
Starzl, T. E., Porter, K. A., Kashiwagi, N. and others. ibid. 1975, 140, 549. 3. Mackenzie, R. J., Leiberman, D. P., Mathie, R. T., Rice, G. C., Harper, A. M., Blumgart, L. H. Acta chir. scand. (in the press). 4. Schneider, W. C., Greco, A. Biochim. biophys. Acta, 1971, 228, 610. 5. Bucher, N. L. R., Swaffield, M. N. Cancer Res. 1973, 33, 3189. 6. Report of a Joint Working Party of the Royal College of Physicians of London and the British Cardiac Society Jl R. Coll. Physns. 1976, 10, 213. 7. ibid. 1975, 10, 5.