436
apparently successful therapy is strongly suggestive of relapse. In our experience about a fifth of patients who subsequently relapse will have abnormal titres of before there is other evidence of disease A activity. high titre after initial therapy is a strong indication for thorough investigations, again with particular serum-A.F.p.
lung and supradiaphragmatic nodal metasregard tases. Finally, a normal titre does not necessarily exclude the presence of tumour, although these observations suggest that when the initial titre is high and falls to normal levels after radiotherapy this may indicate that nodal metastases have been eradicated. However, we found that 27% of tumours produced abnormal amounts of A.F.P. at one time and failed to do so at another, so that a normal A.F.P. titre in a patient in whom the serum-A.F.p. was raised before treatment does not exclude disease activity. There can be no doubt that measurement of the serum-A.F.p. is clinically useful in the management of patients with testicular tumours. A raised serum-A.F.p. titre in a patient after orchidectomy, confirmed by repeated examination, should be regarded as definite proof of residual tumour so long as there is no obvious liver disease. It is unfortunate that in this series only 1 patient had a pre-orchidectomy A.F.P. titre estimated. Clearly this should now be part of the clinical management of any patient in whom a testicular tumour is sus-
PROGNOSTIC SIGNIFICANCE OF IN-VITRO COMPLEMENT FIXATION IN LIVER BIOPSY SPECIMENS FROM PATIENTS WITH ACUTE VIRAL HEPATITIS TYPE B
Department of Gastroenterology, Ospedale Mauriziano Umberto I, Corso Turati 46, 10128 Turin, Italy
prognostic significance of in-vitro complement fixation (V.C.F.) by hepatitis-B core antigen/antibody immunocomplexes in hepatitis-B surface antigen (HBsAg) positive liver biopsy specimens was prospectively evaluated in 47 patients presenting with acute viral hepatitis type B. 34 of 37 V.C.F.-negative patients made an uneventful recovery and became HBsAg negative; in all patients with a V.C.F.-positive test chronic hepatitis and persistent antigenæmia developed. The V.C.F. test is a simple and reliable prognostic indicator of persistent infection and of progression of apparently acute hepatitis to a chronic liver disorder.
Introduction
IN:VITRO complement fixation (v.c.F.), presumably by core antigen (HBcAg) immune complexes,
hepatitis-B
We thank the many surgeons and radiotherapists who have referred to the testicular tumour unit. The histology was reviewed in every case by Prof. N. F. C. Gowing or Prof. A. M. Neville, both members of the Testicular Tumour Panel of Great Britain. Radiological investigations were done by Dr J. S. Macdonald. Mrs D. E. Austin provided valuable help with the data, and secretarial help was given by Mrs Jean Lewis.
was
Requests for reprints should be addressed to T.J.McE. REFERENCES
Abelev, G. I., Assercritova, I, V., Kraevsky, N. A., Perova, S. D., Perevodchikova, N. I. Cancer, 1967, 2, 551. 2. Abelev, G. I. Cancer Res. 1971, 14, 295. 3. Masopust, J., Kithier, K., Radl, J., Koutecky, J., Kotal, L. Int. J. Cancer, 1968, 3, 364. 4. Hull, E. W., Moertel, C. G., Carbone, P. P. Clin. Res. 1969, 17, 403. 5. Adinolfi, A., Adinolfi, M., Lessof, M. H. J. med. Genet. 1975, 12, 138. 6. Mawas, C., Kohen, M., Lemerle, J., Buffe, D., Schweisguth, O., Burtin, P. Int. J. Cancer, 1969, 4, 76. 7. Lamerz, R., Fateh Moghadam, A. Klin. Wschr. 1975, 53, 147. 8. Bourgeaux, G., Martin, F., Aupecle, P., Guerrin, J. Presse méd. 1971, 79, 1.
1589. D. A., Abelev, G. I., Levina, D. M., Marienbach, E. V., Martochkina, G. A., Laskina, A. V., Solovjeva, E. A. Int. J. Cancer, 1973, 11, 586. 10. Buffe, D. GANN Monogr. Cancer Res. 1973, 14, 117. 11. Mawas, C., Buffe, D., Schweisguth, O., Burtin, P. Rev. Eur. clin. Biol. 1971, 16, 430. 12. Alpert, E., Hersberg, R., Schur, P. H., Isselbacher, K. J. Gastroenterology, 1971, 61, 137. 13. Collins, D. H., Pubh, R. C. B. Br. J. Urol. 1964, 36, suppl. 2. 14. Peckham, M. J., McElwain, T. J. Clins Endocr. Metab. 1975, 4, 665. 15. Kohn, J., Kahan, M. J. immun. Meth. 1976, 11, 303. 16. Purves, L. R., Purves, M. S. Afr. med. J. 1972, 46, 1290. 17. Nishi, S., Hirai, H. GANN Monogr. Cancer Res. 1973, 14, 79. 18. Leek, A. E., Ruoss, C. F., Kitau, M. J., Chard, T. Br. J. Obstet. Gynaec. 1975, 82, 669. 19. Masseyef, R. Path. Bil. 1972, 20, 703. 20. Ruoslahti, E., Seppala, M. Nature, New Biol. 1972, 235, 161. 21. Hirai, H., Nishi, S., Watabe, H. J. nucl. Med. 1973, 13, 783. 22. Monod, J., Jacob, F. Symp. quant. Biol. 1961, 26, 193. 23. Omenn, G. S. Ann. intern. Med. 1970, 72, 136. 24. Ballas, M. Ann. clin. Lab. Sci. 1974, 4, 267. 25. Tsuchida, Y., Endo, Y., Urano, Y., Ishida, M. Ann. N.Y. Acad. Sci. 1975, 9.
Elgort,
1, 221. 26. Talerman, A., Haije, W. G. Cancer, 1974, 34, 1722. 27. Grigor, K., Neville, A. M. Personal communication. G. Acta path. microbiol. scand. 1975, 83, 573. 29. Palmer, P. E., Safaii, H., Wolfe, H. J. Am. J. clin. Path. 1976, 65, 575. 30. Tyrell, C. J., Peckham, M. J. Unpublished.
28.
Norgaard Pedersen, B., Albrechtsen, R., Teilum,
The
Summary
pected. patients
F. BONINO G. VERME
M. RIZZETTO S. DIANA
to
detected in the liver biopsy specimens of patients with chronic hepatitis-B surface antigen (HBsAg) positive liver disease, whereas it was not observed in hepatitis, or in healthy carriers, in acute self-limited HBsAg-negative liver disorders. 12 Since it is often impossible to predict the chronic evolution of apparently acute hepatitis on biochemical and histological grounds, we studied the prognostic significance of v.c.F. detection by prospectively evaluating 47 patients with no evidence of previous liver disease, presenting clinically with acute type-B viral hepatitis. The results of the V.C.F. test in the biopsy specimens taken after admission to hospital and the appearance of symptoms were correlated with the histological and clinical data at follow-up 7-12 months afterwards, Persistence of antigenaemia and the development of chronic liver disease were strikingly more common in the
v.c.F.-positive patients. Patients and Methods 47 patients with no evidence of previous liver disease, pre senting with an episode of acute HBsAg-positive hepatitis, were studied. Patients with a history of jaundice or hepatin or with abnormal serum-protein concentrations were exclude The clinical diagnosis of acute type-B hepatitis was basedoc the
recent onset
of symptoms, abnormal concentrationsc:
serum-transaminases, and detection of HBsAg by radio munoassay (R.I.A.). Liver biopsy specimens were obtainr.’ within 30 days of admission to hospital, usually between tbw 4th and 9th days. Steroids or azathioprine were not given any patient. After leaving hospital all patients were followed up for to a
year
at
two-monthly intervals by physical examination;
terviews; determinations of serum bilirubin, transaminas and protein concentrations; and detection of circuit HBsAg by R.I.A. A second biopsy specimen was obtained from all patiend who still had symptoms 7-12 months after the first bior-
437 TABLE
1—HISTOLOGICAL, IMMUNOLOGICAL
AND BIOCHEMICAL DATA IN
37
V.C.F-NEGATIVE PATIENTS PRESENTING CLINICALLY WITH ACUTE TYPE-B
HEPATITIS ON ADMISSION TO HOSPITAL AND AFTER
G.o GT
MONTHS AFTER ONSET OF SYMPTOMS
L=;erum-glutamic-{)xaloacetic-transaminose. =serum-glutamic-pyruvic-transaminase
T
Only a few symptom-free patients consented to a second biopsy ,table i). A part of each liver specimen was immediately frozen; the rest was fixed in 10% formalin and stained with haematoxylin and eosin, with van Gieson, with silver reticulin stain and with a modified trichrome stain.’ The histological criteria used were 4
those of de Groote et al.’ Cryostat sections of each biopsy specimen, fixed in ether for ! minutes, were stained with antisera against HBsAg and HBcAg, human IgG, C3, and Clq and were studied by immunofluorescence (I.F.L.) for their capacity to bind human complement in vitro. The preparation and specificities of HBs and HBc antisera and the details of the v.c.F. test have been reported elsewhere.’5 antisera against human IgG, C3, and Clq were obtained commercially (Behringwerke).
Results
detected in 10 biopsy specinens. The histological, immunological, and biochemical data of 37 patients with a negative v.c.F. test are summarised in table I, while the data of the patients with a positive v.c.F. test v.c.F. was
are
7-12
reported in table n.
V,C.F.-negative Patients In the v.c.F.-negative group (table i) histology showed acute hepatitis in 21 patients and a chronic active hepatitis in 3. There were 13 biopsy specimens with a variable combination of parenchymal necrosis, portal and lobular inflammation, reticulin collapse and discrete early fibrosis, on which 3 experienced pathologists with no knowledge of the clinical history could not agree, the picture being compatible either with a prolonged acute hepatitis or with early active chronic disease. Since there was no anamnestic or biochemical evidence of previous liver disease, acute hepatitis running a protracted course was provisionally diagnosed in these patients. At follow-up, transaminase concentrations were normal and HBsAg had been cleared from the blood in all V.C.F.-negative patients with acute and acute prolonged hepatitis; none of them had symptoms. 8 v.c.F.-negative patients gave their consent to a second biopsy between 7 and 12 months after the pre:ious one; the liver was normal in each case, except for a few lobular mononuclear infiltrates in a patient in
prolonged hepatitis had been diagnosed. Abnormal serum-enzyme concentrations and HBs antigenæmia persisted in each of the 3 v.c.F.-negative pa-
*hom
acute
tients with evidence of chronic active hepatitis in their
biopsy specimen; examination of a second liver specimen confirmed the negative i.F.L. test and the histological findings observed in the first biopsy. HBsAg and HBcAg were seldom detected in the liver sections in this group; there were no intranuclear IgG deposits in any biopsy specimen. initial
V.C.F.-positive Patients In the v.c.F.-positive group (table II) chronic active and chronic persistent hepatitis was already established in 2 patients by examination of the first biopsy specimen ; acute hepatitis was observed in 2 patients; and 6 had acute prolonged hepatitis indistinguishable from the histological picture seen in the v.c.F.-negative group. 2 patients were anicteric. Although serum-transaminases started to decline promptly, they remained abnormal and HBsAg was always detected in each serum specimen collected during follow-up. A chronic histological lesion was found in each biopsy specimen obtained between the 7th and the 12th month- after symptoms appeared. The histological diagnosis reached on the basis of the first and second biopsy specimen from each v.c.F.-positive patient is reported in table n. v.c.F. occurred in all the follow-up liver specimens. HBsAg was detected in the cytoplasm of a few specimens, HBcAg was stained by I.F.L. in all v.c.F.-positive biopsy specimens except the second one from patient 2 (table n) which fixed complement and reacted with IgG but not with HBc antiserum; core particles were, however, detected in great quantity in this case by electron microscopy. 9 patients had symptoms during the fol-
low-up period. Discussion All v.c.F.-negative patients with acute or acute prolonged hepatitis had normal serum-enzymes and no longer had circulating HBsAg after one year, antigenæmia disappearing usually within three months; they all remained symptom-free. Only a few patients in this group consented to a second biopsy which in each case showed a normal liver or only slight abnormalities. In contrast, serum-enzyme concentrations remained
constantly abnormal and HBsAg persisted in the blood of all v.c.F.-positive patients during follow-up. Biochemical studies could not predict persistence of infec-
438 TABLE
II-HISTOLOGICAL, IMMUNOLOGICAL,
10
AND BIOCHEMICAL DATA IN
VIRAL HEPATITIS AT ADMISSION TO HOSPITAL AND
V.C.F.-POSITIVE PATIENTS PRESENTING CLINICALLY WITH ACUTE
7-12 MONTHS,
chronic active active cirrhosis. *anicteric.
A.H.= acute
C.A.H.=
A.P.H.= acute
A.c.=
hepatitis. prolonged hepatitis. C.P.H.= chronic persistent hepatitis.
AFTER ONSET OF SYMPTOMS
tion or the chronic course of the disease; mean serum bilirubin and transaminase concentrations during the acute episode were lower in the v.c.F.-positive group than in the v.c.F.-negative group but were of no prognostic significance in the individual patient. Although in 2 v.c.F.positive patients a chronic lesion was established by examination of the first biopsy specimen, in the remaining cases the tendency to chronicity remained uncertain. In these biopsy specimens, a variable combination of necrosis, inflammation, reticulin collapse, and early fibrosis could be interpreted either as acute hepatitis running a protracted course or as early chronic active disease; identical histological pictures were observed in the v.c.F.-negative group. The provisional diagnosis of protracted hepatitis was confirmed in all v.c.F.-negative cases at follow-up while chronic hepatitis was diagnosed in all the v.c.F.-positive cases.
The v.c.F. test, therefore, appeared to us to be the single most reliable test for the identification of patients likely to have persistent infection and chronic HBsAg liver disease. The few patients with chronic disease and persistent antigenaemia despite a negative v.c.F. test might represent either different pathogenetical entities or sampling errors due to a low and uneven distribution of infected hepatocytes. It must be stressed, however, that a positive v.c.F. test did not imply active disease nor progression to nodular regeneration; although all v.c.F.-positive patients had a chronic lesion in the second biopsy specimen, it varied from persistent hepatitis to aggressive cirrhosis, irrespective of the extent and strength of v.c.F. immunofluorescence. The follow-up period is too short to exclude a cirrhotic outcome of the disease in these subject; however, in 2 patients studied for two years, scarring and a decrease in portal infiltrates were noted in the last biopsy specimen and no evidence of nodular transformation was found at laparoscopy, despite a persistent positive V.C.F. test.
Circumstantial evidence suggests that complement is fixed by HBc antigen/antibody complexes.l2 Complement binding never occurred in HBsAg-negative disorders, while in previous studies HBc has been always detected in v.c.F.-positive biopsy specimens.l2 IgG de-
hepatitis.
posits have repeatedly been noted in liver-cell nuclei containing HBc determinant.12 67 In this study only one v.c.F.-positive sample, the second biopsy specimen from patient 2 (table n), did not with HBc antiserum. We have also seen strong with minimal HBc fluorescence in liver specimens from patients with advanced cirrhosis, and we interpreted this as the masking of HBc determinants by excess antibody fixed in vivo which allowed binding of complement but not of specific HBc fluorescent antiserum.The finding by electron microscopy of core particles in patient 2 supports this hypothesis and suggests that v.c.F. must be tested for before the presence of HBc in a biopsy specimen is excluded because of a negativc result by direct I.F.L. The v.c.F. test is sensitive and simple to perform; it can be carried out with commercially available antisera against complement components. Our results lead to important questions about therapy in v.c.F.-positive patients. If v.C.F.-positive patients are more at risk of persistent infection and chronic disease should they be treated irrespective of a histological diagnosis of acute or chronic persistent hepatitisbenign conditions in which therapy is contraindicated Is there any rationale in treating v.c.F.-positive patients with conventional immunosuppressive drugs, when the nature of the immunological response described is unknown and might even be the expression of an hypoergic immunological system? Further experimental studies and controlled trials are needed to answer these ques react
v.c.F.
tions. We thank Dr M. G. Canese for electron
microscopy in patient 2.
Requests for reprints should be addressed to M.R. REFERENCES 1.
Rizzetto, M., Bonino, F., Crivelli, O., Canese, M. G., Verme, G. Gut (in the
press). 2. Gerber, M. A., Sarno,
E., Vernace, S. V. New Engl. J. Med. 1976, 294,
922. 3. Gubetta, L., Rizzetto, M., Verme, G. Pathologica (in the press). Groote, J., Desmet, V. J., Gedick, P., Korb, G., Popper, H., Poulsen, H, Schever, P. J., Schmid, M., Thaler, H., Uehlinger, E., Wepler, W. Lancet, 1968, ii, 626. 5. Rizzetto, M., Crivelli, O., Suriani, R., Verme, G. Ricerca Clinica Lab, 1976,
4. De
6, 41. 6. 7.
Hadziyannis, S. Digestion, 1973, 8, 437. Arnold, W., Meyer zum Buschenfelde, K. H., Hess, G., Knolle, J.ibid. 1975, 12, 277.
apparently successful therapy is strongly suggestive of relapse. In our experience about a fifth of patients who subsequently relapse will have abnormal titres of before there is other evidence of disease A activity. high titre after initial therapy is a strong indication for thorough investigations, again with particular serum-A.F.p.
lung and supradiaphragmatic nodal metasregard tases. Finally, a normal titre does not necessarily exclude the presence of tumour, although these observations suggest that when the initial titre is high and falls to normal levels after radiotherapy this may indicate that nodal metastases have been eradicated. However, we found that 27% of tumours produced abnormal amounts of A.F.P. at one time and failed to do so at another, so that a normal A.F.P. titre in a patient in whom the serum-A.F.p. was raised before treatment does not exclude disease activity. There can be no doubt that measurement of the serum-A.F.p. is clinically useful in the management of patients with testicular tumours. A raised serum-A.F.p. titre in a patient after orchidectomy, confirmed by repeated examination, should be regarded as definite proof of residual tumour so long as there is no obvious liver disease. It is unfortunate that in this series only 1 patient had a pre-orchidectomy A.F.P. titre estimated. Clearly this should now be part of the clinical management of any patient in whom a testicular tumour is sus-
PROGNOSTIC SIGNIFICANCE OF IN-VITRO COMPLEMENT FIXATION IN LIVER BIOPSY SPECIMENS FROM PATIENTS WITH ACUTE VIRAL HEPATITIS TYPE B
Department of Gastroenterology, Ospedale Mauriziano Umberto I, Corso Turati 46, 10128 Turin, Italy
prognostic significance of in-vitro complement fixation (V.C.F.) by hepatitis-B core antigen/antibody immunocomplexes in hepatitis-B surface antigen (HBsAg) positive liver biopsy specimens was prospectively evaluated in 47 patients presenting with acute viral hepatitis type B. 34 of 37 V.C.F.-negative patients made an uneventful recovery and became HBsAg negative; in all patients with a V.C.F.-positive test chronic hepatitis and persistent antigenæmia developed. The V.C.F. test is a simple and reliable prognostic indicator of persistent infection and of progression of apparently acute hepatitis to a chronic liver disorder.
Introduction
IN:VITRO complement fixation (v.c.F.), presumably by core antigen (HBcAg) immune complexes,
hepatitis-B
We thank the many surgeons and radiotherapists who have referred to the testicular tumour unit. The histology was reviewed in every case by Prof. N. F. C. Gowing or Prof. A. M. Neville, both members of the Testicular Tumour Panel of Great Britain. Radiological investigations were done by Dr J. S. Macdonald. Mrs D. E. Austin provided valuable help with the data, and secretarial help was given by Mrs Jean Lewis.
was
Requests for reprints should be addressed to T.J.McE. REFERENCES
Abelev, G. I., Assercritova, I, V., Kraevsky, N. A., Perova, S. D., Perevodchikova, N. I. Cancer, 1967, 2, 551. 2. Abelev, G. I. Cancer Res. 1971, 14, 295. 3. Masopust, J., Kithier, K., Radl, J., Koutecky, J., Kotal, L. Int. J. Cancer, 1968, 3, 364. 4. Hull, E. W., Moertel, C. G., Carbone, P. P. Clin. Res. 1969, 17, 403. 5. Adinolfi, A., Adinolfi, M., Lessof, M. H. J. med. Genet. 1975, 12, 138. 6. Mawas, C., Kohen, M., Lemerle, J., Buffe, D., Schweisguth, O., Burtin, P. Int. J. Cancer, 1969, 4, 76. 7. Lamerz, R., Fateh Moghadam, A. Klin. Wschr. 1975, 53, 147. 8. Bourgeaux, G., Martin, F., Aupecle, P., Guerrin, J. Presse méd. 1971, 79, 1.
1589. D. A., Abelev, G. I., Levina, D. M., Marienbach, E. V., Martochkina, G. A., Laskina, A. V., Solovjeva, E. A. Int. J. Cancer, 1973, 11, 586. 10. Buffe, D. GANN Monogr. Cancer Res. 1973, 14, 117. 11. Mawas, C., Buffe, D., Schweisguth, O., Burtin, P. Rev. Eur. clin. Biol. 1971, 16, 430. 12. Alpert, E., Hersberg, R., Schur, P. H., Isselbacher, K. J. Gastroenterology, 1971, 61, 137. 13. Collins, D. H., Pubh, R. C. B. Br. J. Urol. 1964, 36, suppl. 2. 14. Peckham, M. J., McElwain, T. J. Clins Endocr. Metab. 1975, 4, 665. 15. Kohn, J., Kahan, M. J. immun. Meth. 1976, 11, 303. 16. Purves, L. R., Purves, M. S. Afr. med. J. 1972, 46, 1290. 17. Nishi, S., Hirai, H. GANN Monogr. Cancer Res. 1973, 14, 79. 18. Leek, A. E., Ruoss, C. F., Kitau, M. J., Chard, T. Br. J. Obstet. Gynaec. 1975, 82, 669. 19. Masseyef, R. Path. Bil. 1972, 20, 703. 20. Ruoslahti, E., Seppala, M. Nature, New Biol. 1972, 235, 161. 21. Hirai, H., Nishi, S., Watabe, H. J. nucl. Med. 1973, 13, 783. 22. Monod, J., Jacob, F. Symp. quant. Biol. 1961, 26, 193. 23. Omenn, G. S. Ann. intern. Med. 1970, 72, 136. 24. Ballas, M. Ann. clin. Lab. Sci. 1974, 4, 267. 25. Tsuchida, Y., Endo, Y., Urano, Y., Ishida, M. Ann. N.Y. Acad. Sci. 1975, 9.
Elgort,
1, 221. 26. Talerman, A., Haije, W. G. Cancer, 1974, 34, 1722. 27. Grigor, K., Neville, A. M. Personal communication. G. Acta path. microbiol. scand. 1975, 83, 573. 29. Palmer, P. E., Safaii, H., Wolfe, H. J. Am. J. clin. Path. 1976, 65, 575. 30. Tyrell, C. J., Peckham, M. J. Unpublished.
28.
Norgaard Pedersen, B., Albrechtsen, R., Teilum,
The
Summary
pected. patients
F. BONINO G. VERME
M. RIZZETTO S. DIANA
to
detected in the liver biopsy specimens of patients with chronic hepatitis-B surface antigen (HBsAg) positive liver disease, whereas it was not observed in hepatitis, or in healthy carriers, in acute self-limited HBsAg-negative liver disorders. 12 Since it is often impossible to predict the chronic evolution of apparently acute hepatitis on biochemical and histological grounds, we studied the prognostic significance of v.c.F. detection by prospectively evaluating 47 patients with no evidence of previous liver disease, presenting clinically with acute type-B viral hepatitis. The results of the V.C.F. test in the biopsy specimens taken after admission to hospital and the appearance of symptoms were correlated with the histological and clinical data at follow-up 7-12 months afterwards, Persistence of antigenaemia and the development of chronic liver disease were strikingly more common in the
v.c.F.-positive patients. Patients and Methods 47 patients with no evidence of previous liver disease, pre senting with an episode of acute HBsAg-positive hepatitis, were studied. Patients with a history of jaundice or hepatin or with abnormal serum-protein concentrations were exclude The clinical diagnosis of acute type-B hepatitis was basedoc the
recent onset
of symptoms, abnormal concentrationsc:
serum-transaminases, and detection of HBsAg by radio munoassay (R.I.A.). Liver biopsy specimens were obtainr.’ within 30 days of admission to hospital, usually between tbw 4th and 9th days. Steroids or azathioprine were not given any patient. After leaving hospital all patients were followed up for to a
year
at
two-monthly intervals by physical examination;
terviews; determinations of serum bilirubin, transaminas and protein concentrations; and detection of circuit HBsAg by R.I.A. A second biopsy specimen was obtained from all patiend who still had symptoms 7-12 months after the first bior-
437 TABLE
1—HISTOLOGICAL, IMMUNOLOGICAL
AND BIOCHEMICAL DATA IN
37
V.C.F-NEGATIVE PATIENTS PRESENTING CLINICALLY WITH ACUTE TYPE-B
HEPATITIS ON ADMISSION TO HOSPITAL AND AFTER
G.o GT
MONTHS AFTER ONSET OF SYMPTOMS
L=;erum-glutamic-{)xaloacetic-transaminose. =serum-glutamic-pyruvic-transaminase
T
Only a few symptom-free patients consented to a second biopsy ,table i). A part of each liver specimen was immediately frozen; the rest was fixed in 10% formalin and stained with haematoxylin and eosin, with van Gieson, with silver reticulin stain and with a modified trichrome stain.’ The histological criteria used were 4
those of de Groote et al.’ Cryostat sections of each biopsy specimen, fixed in ether for ! minutes, were stained with antisera against HBsAg and HBcAg, human IgG, C3, and Clq and were studied by immunofluorescence (I.F.L.) for their capacity to bind human complement in vitro. The preparation and specificities of HBs and HBc antisera and the details of the v.c.F. test have been reported elsewhere.’5 antisera against human IgG, C3, and Clq were obtained commercially (Behringwerke).
Results
detected in 10 biopsy specinens. The histological, immunological, and biochemical data of 37 patients with a negative v.c.F. test are summarised in table I, while the data of the patients with a positive v.c.F. test v.c.F. was
are
7-12
reported in table n.
V,C.F.-negative Patients In the v.c.F.-negative group (table i) histology showed acute hepatitis in 21 patients and a chronic active hepatitis in 3. There were 13 biopsy specimens with a variable combination of parenchymal necrosis, portal and lobular inflammation, reticulin collapse and discrete early fibrosis, on which 3 experienced pathologists with no knowledge of the clinical history could not agree, the picture being compatible either with a prolonged acute hepatitis or with early active chronic disease. Since there was no anamnestic or biochemical evidence of previous liver disease, acute hepatitis running a protracted course was provisionally diagnosed in these patients. At follow-up, transaminase concentrations were normal and HBsAg had been cleared from the blood in all V.C.F.-negative patients with acute and acute prolonged hepatitis; none of them had symptoms. 8 v.c.F.-negative patients gave their consent to a second biopsy between 7 and 12 months after the pre:ious one; the liver was normal in each case, except for a few lobular mononuclear infiltrates in a patient in
prolonged hepatitis had been diagnosed. Abnormal serum-enzyme concentrations and HBs antigenæmia persisted in each of the 3 v.c.F.-negative pa-
*hom
acute
tients with evidence of chronic active hepatitis in their
biopsy specimen; examination of a second liver specimen confirmed the negative i.F.L. test and the histological findings observed in the first biopsy. HBsAg and HBcAg were seldom detected in the liver sections in this group; there were no intranuclear IgG deposits in any biopsy specimen. initial
V.C.F.-positive Patients In the v.c.F.-positive group (table II) chronic active and chronic persistent hepatitis was already established in 2 patients by examination of the first biopsy specimen ; acute hepatitis was observed in 2 patients; and 6 had acute prolonged hepatitis indistinguishable from the histological picture seen in the v.c.F.-negative group. 2 patients were anicteric. Although serum-transaminases started to decline promptly, they remained abnormal and HBsAg was always detected in each serum specimen collected during follow-up. A chronic histological lesion was found in each biopsy specimen obtained between the 7th and the 12th month- after symptoms appeared. The histological diagnosis reached on the basis of the first and second biopsy specimen from each v.c.F.-positive patient is reported in table n. v.c.F. occurred in all the follow-up liver specimens. HBsAg was detected in the cytoplasm of a few specimens, HBcAg was stained by I.F.L. in all v.c.F.-positive biopsy specimens except the second one from patient 2 (table n) which fixed complement and reacted with IgG but not with HBc antiserum; core particles were, however, detected in great quantity in this case by electron microscopy. 9 patients had symptoms during the fol-
low-up period. Discussion All v.c.F.-negative patients with acute or acute prolonged hepatitis had normal serum-enzymes and no longer had circulating HBsAg after one year, antigenæmia disappearing usually within three months; they all remained symptom-free. Only a few patients in this group consented to a second biopsy which in each case showed a normal liver or only slight abnormalities. In contrast, serum-enzyme concentrations remained
constantly abnormal and HBsAg persisted in the blood of all v.c.F.-positive patients during follow-up. Biochemical studies could not predict persistence of infec-
438 TABLE
II-HISTOLOGICAL, IMMUNOLOGICAL,
10
AND BIOCHEMICAL DATA IN
VIRAL HEPATITIS AT ADMISSION TO HOSPITAL AND
V.C.F.-POSITIVE PATIENTS PRESENTING CLINICALLY WITH ACUTE
7-12 MONTHS,
chronic active active cirrhosis. *anicteric.
A.H.= acute
C.A.H.=
A.P.H.= acute
A.c.=
hepatitis. prolonged hepatitis. C.P.H.= chronic persistent hepatitis.
AFTER ONSET OF SYMPTOMS
tion or the chronic course of the disease; mean serum bilirubin and transaminase concentrations during the acute episode were lower in the v.c.F.-positive group than in the v.c.F.-negative group but were of no prognostic significance in the individual patient. Although in 2 v.c.F.positive patients a chronic lesion was established by examination of the first biopsy specimen, in the remaining cases the tendency to chronicity remained uncertain. In these biopsy specimens, a variable combination of necrosis, inflammation, reticulin collapse, and early fibrosis could be interpreted either as acute hepatitis running a protracted course or as early chronic active disease; identical histological pictures were observed in the v.c.F.-negative group. The provisional diagnosis of protracted hepatitis was confirmed in all v.c.F.-negative cases at follow-up while chronic hepatitis was diagnosed in all the v.c.F.-positive cases.
The v.c.F. test, therefore, appeared to us to be the single most reliable test for the identification of patients likely to have persistent infection and chronic HBsAg liver disease. The few patients with chronic disease and persistent antigenaemia despite a negative v.c.F. test might represent either different pathogenetical entities or sampling errors due to a low and uneven distribution of infected hepatocytes. It must be stressed, however, that a positive v.c.F. test did not imply active disease nor progression to nodular regeneration; although all v.c.F.-positive patients had a chronic lesion in the second biopsy specimen, it varied from persistent hepatitis to aggressive cirrhosis, irrespective of the extent and strength of v.c.F. immunofluorescence. The follow-up period is too short to exclude a cirrhotic outcome of the disease in these subject; however, in 2 patients studied for two years, scarring and a decrease in portal infiltrates were noted in the last biopsy specimen and no evidence of nodular transformation was found at laparoscopy, despite a persistent positive V.C.F. test.
Circumstantial evidence suggests that complement is fixed by HBc antigen/antibody complexes.l2 Complement binding never occurred in HBsAg-negative disorders, while in previous studies HBc has been always detected in v.c.F.-positive biopsy specimens.l2 IgG de-
hepatitis.
posits have repeatedly been noted in liver-cell nuclei containing HBc determinant.12 67 In this study only one v.c.F.-positive sample, the second biopsy specimen from patient 2 (table n), did not with HBc antiserum. We have also seen strong with minimal HBc fluorescence in liver specimens from patients with advanced cirrhosis, and we interpreted this as the masking of HBc determinants by excess antibody fixed in vivo which allowed binding of complement but not of specific HBc fluorescent antiserum.The finding by electron microscopy of core particles in patient 2 supports this hypothesis and suggests that v.c.F. must be tested for before the presence of HBc in a biopsy specimen is excluded because of a negativc result by direct I.F.L. The v.c.F. test is sensitive and simple to perform; it can be carried out with commercially available antisera against complement components. Our results lead to important questions about therapy in v.c.F.-positive patients. If v.C.F.-positive patients are more at risk of persistent infection and chronic disease should they be treated irrespective of a histological diagnosis of acute or chronic persistent hepatitisbenign conditions in which therapy is contraindicated Is there any rationale in treating v.c.F.-positive patients with conventional immunosuppressive drugs, when the nature of the immunological response described is unknown and might even be the expression of an hypoergic immunological system? Further experimental studies and controlled trials are needed to answer these ques react
v.c.F.
tions. We thank Dr M. G. Canese for electron
microscopy in patient 2.
Requests for reprints should be addressed to M.R. REFERENCES 1.
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