- Email: [email protected]
TYPE III PROCOLLAGEN PEPTIDE: A MARKER OF DISEASE ACTIVITY AND PROGNOSIS IN PRIMARY BILIARY CIRRHOSIS
1021
should allow logical treatment for the disabling postural hypotension that usually accompanies autonomic failure. For example, in patients with "flat" noradrenaline responses to edrophonium it would be inappropriate to use a drug that stimulates noradrenaline release.
Furthermore, results of such
a test
We thank Dr R. J. Prescott, Medical Computing and Statistics Unit, University of Edinburgh for his help with the statistical analysis; Dr R. A. Riemersma, Cardiovascular Research Unit, University of Edinburgh, for his help with the measurements of plasma noradrenaline; and Dr P. Matute for his assistance with the studies themselves. The study was supported by a
grant from the Wellcome Trust.
Correspondence should be addressed to D. J. E., Department of Medicine, Royal Infirmary, Edinburgh EH3 9YW. REFERENCES 1 Bannister R. Introduction and classification In Bannister R, ed Autonomic failure. Oxford- Oxford University Press, 1983 1-13
TYPE III PROCOLLAGEN PEPTIDE: A MARKER OF DISEASE ACTIVITY AND PROGNOSIS IN PRIMARY BILIARY CIRRHOSIS
CHRISTOPHER BABBS1 LINDA P. HUNT2 NAJIB Y. HABOUBI3
ALEXANDER SMITH1 BRENDA P. ROWAN1 THOMAS W. WARNES1
Liver Unit, University Department of Gastroenterology,1 and Faculty of Medicine Computational Group,2 Manchester Royal Infirmary; and Department of Pathology, University Hospital of South Manchester3
The prognostic value of the aminoterminal propeptide of type III procollagen (P3NP) was investigated in 63 patients with primary biliary cirrhosis (PBC) followed for up to 87 months. No patient with an initially normal serum P3NP level died during the study; survival was significantly worse with increasing serum P3NP levels. Cox multivariate analysis confirmed that serum P3NP was an independent prognostic variable. Positive correlations were found between serum P3NP and histological stage, pericellular fibrosis, piecemeal necrosis, and serum concentrations of alanine aminotransferase and aspartate aminotransferase. Raised P3NP levels also correlated with the degree of cholestasis as evaluated by serum bilirubin, serum alkaline phosphatase, and copper binding protein deposition in the liver. Serum P3NP is of prognostic value because it reflects the major pathophysiological features of PBC.
Summary
JG, Tuck RR. Disorders of the autonomic nervous system. part 1. Pathophysiology and clinical features. Ann Neurol 1987, 21: 419-30. 3 Oppenheimer D Neuropathology of progressive autonomic failure. In. Bannister R, ed Autonomic failure. Oxford: Oxford University Press, 1983; 267-83. 4. Leveston SA, Shah SD, Cryer PE Cholinergic stimulation of norepinephrine release in man. Evidence of a sympathetic postganglionic axonal lesion in diabetic adrenergic neuropathy. J Clin Invest 1979, 64: 374-80 5. Ewing DJ, Bellavere F, Espi F, et al. Correlation of cardiovascular and neuroendocrine tests of autonomic function in diabetes. Metabolism 1986, 35: 349-53. 6. Matthews DM, Martyn CN, Riemersma RA, Clarke BF, Ewing DJ. Noradrenaline response to edrophonium (Tensilon) and its relation to other autonomic tests in diabetic subjects Diabetes Res 1987, 6: 175-80 7. Ewing DJ, Martyn CN, Young RJ, Clarke BF The value of cardiovascular autonomic function tests 10 years’ experience in diabetes Diabetes Care 1985; 8: 491-98. 8. Da Prada M, Zurcher G Simultaneous radioenzymatic determination of plasma and tissue adrenaline, noradrenaline and dopamine within the femtomole range. Life Sci 1976, 19: 1161-73. 9. Taylor P. Anticholinesterase agents In Gilman AG, Goodman LS, Rall TW, Murad F, eds Goodman and Gilman’s pharmacological basis of therapeutics, 7th ed New York Macmillan, 1985 110-29 10. Sever PS Plasma noradrenaline in autonomic failure. In: Bannister R, ed. Autonomic failure Oxford: Oxford University Press, 1983; 155-73 11. Polinsky RJ. Pharmacological responses and biochemical changes in progressive autonomic failure. In. Bannister R, ed. Autonomic failure. Oxford: Oxford University Press, 1983:201-36. 2 McLeod
the diagnosis and follow-up of patients with acute and chronic liver disease9-13 and in the assessment of disease activity in autoimmune chronic active hepatitis.14 The interpretation of raised serum P3NP concentrations in PBC has been complicated by the small number of patients studied;9,15-17 one report suggests that the test may be of prognostic value.155 The aim of the present study was to examine the prognostic value of serum P3NP in a large group of PBC patients and to compare it with other factors previously shown to influence survival. In addition, we wished to test our hypothesis that this assay might reflect overall disease activity by relating serum levels to the histological stage of the disease and the degree of fibrosis, as well as to markers of necrosis, inflammation, and cholestasis.
Patients and Methods The study group consisted of 63 patients (57 female; mean age 57-8 years, range 29-78) prospectively evaluated in our unit before entry into a controlled trial of colchicine therapy’8 and followed for a mean of 37 months (range 0 6—87). PBC was diagnosed by conventional criteria’9 and patients were divided into those with
typical symptoms (pruritus, pigmentation, jaundice, ascites, or gastrointestinal bleeding; n = 28) and those who were presymptomatic (n — 35). The colchicine and placebo groups were well matched at entry for all measured indices (table I). There were 22 deaths during the study and in 19 cases this was the result of liver disease. 3 patients died of unrelated causes (1 carcinoma rectum; 1 aplastic anaemia; 1 pneumonia) and these cases were censored from the survival analysis.
Introduction WITH the advent of liver transplantation as an established treatment for patients with end-stage primary biliary cirrhosis (PBC), the assessment of prognosis has become of great clinical importance.’Serum bilirubin has consistently been shown to be an important prognostic guide in PBC;1,3-6 however, the disease is clinically heterogeneous and assessment of prognosis cannot be based on measurement of a single variable.7 The prognostic value of other clinical, biochemical, and histological indices remains controversial. The aminoterminal propeptide of type III procollagen (P3NP) is released during the extracellular processing of type III collagen, a major component of the hepatic extracellular matrix, and may be measured in serum by radioimmunoassay.8 The test has proved of clinical value in
TABLE I-GROUP COMPARABILITY
*Geometnc mean (range) for tArithmetric mean (SD).
log transformed
data.
1022 TABLE II—UNIVARIATE ANALYSIS
Results The upper limit of normal for serum P3NP (mean + 2 SD) was 13 8 ng/ml and concentrations exceeded this in 73% (46/63) of PBC patients (p < 0 001, fig 1). The effect on survival, of the serum P3NP level at entry to the study, is shown in fig 2. No deaths from liver disease were recorded among the 17 patients with initially normal serum P3NP values (group A) although 9 of these patients had bridging fibrosis or cirrhosis (Ludwig stages 3 and 4, fig 3) and 5 had a raised serum bilirubin. The difference in cumulative survival between the three groups was highly significant
(p < 0001).
Effect of different variables survival curves (logrank test).
on
survival estimated from
Kaplan-Meier
Serum was separated soon after collection and stored at - 20°C until assay. P3NP levels were measured by radioimmunoassay (Behringwerke Hoechst, Frankfurt) as described by Rohde et al. 8 A normal reference range was established in our laboratory in 65 healthy adults. The intra-assay coefficient of variation (CV) was 85 % and the inter-assay CV, 12 % (n 7). Liver biopsy specimens were fixed in formalin and staged according to the criteria of Ludwig et al ;20 10 patients were stage 1, 11stage 2, 17 stage 3, and 25 stage 4 (established cirrhosis). The following histological indices were assessed blindly and graded semiquantitatively 0 to + 3: piecemeal necrosis, parenchymal inflammation, cholestasis, and copper binding protein deposition. Parenchymal pericellular (perisinusoidal) fibrosis was also graded 0 to + 3 after staining with haematoxylin-picrosirius red.21 Conventional liver function tests were done by standard =
autoanalyser techniques. Correlation coefficients were calculated by means of Pearson and Kendall non-parametric tests and two-tailed levels of significance are provided throughout. Logarithmic conversion was used to normalise skewed distribution of serum bilirubin, alkaline phosphatase, and prothrombin index. To estimate the relation between each of the 17 variables assessed and survival we applied the logrank test22 to Kaplan-Meier survival curves after dividing the patients into appropriate subgroups (table II). Those variables with a significant influence on prognosis and those that showed a trend towards significance were entered into a Cox multivariate analysis, the proportional hazards model being used to establish independent significance.23 TABLE III-FINAL COX MODEL
*Exponential transformation of age was performed as previously suggested6 positive value for the regression coefficient indicates an adverse influence on survival and vice versa for a negative value. A coefficient of 0 would imply
A
no
effect
on
survival.
Fig
1—Serum P3NP levels in
a
normal group and PBC
Histograms show the means with standard deviation standard deviations (bars).
In addition to indicated that log
serum
serum
patients.
(solid blocks)
and 2
P3NP levels, univariate analysis bilirubin, log prothrombin index,
symptom status, and serum aspartate aminotransferase all had a significant influence on survival (table II). Age, serum albumin, presence or absence of cirrhosis, and treatment status all showed a trend towards prognostic importance but did not reach statistical significance. These factors were, however, included in the Cox multivariate analysis since variables showing a trend toward significance in a univariate analysis may become significant when other factors are adjusted for. The results of this analysis (table III) confirm the previously recognised prognostic value of serum bilirubin, serum albumin, prothrombin index, age, and symptom status. Serum P3NP remains a highly significant prognostic variable which is independent of the other variables in the model. In contrast, the presence or absence of cirrhosis had no independent influence on survival; treatment with colchicine seemed to improve survival, when other factors were adjusted for.
histological cholestasis,
1023
Discussion The present study clearly demonstrates that serum P3NP is an important predictor of survival in PBC and suggests that prognosis is related to the degree to which P3NP is raised. Thus, none of the patients whose initial serum P3NP level was normal (group A) died, despite the fact that 9 of this group had histologically late stage disease. Outcome was worst in those patients whose initial serum P3NP was above the mean value (group C): 52 % (15/29) of these patients died
Fig 2-Cumulative proportion of patients surviving initial P3NP levels (Kaplan-Meier). < 13-8 ng/ml 19 ng/ml (n = 29).
Group A, C
>
(n
=
17); group B,
13 9-19-9
at
ng/ml (n
=
different
17); group
during follow-up. In previous reports there is lack of agreement on the prognostic value of factors other than serum bilirubin 4-6 In the present study, using the Cox proportional hazards model, we have shown that older patients, those presenting with symptoms, and those who have
a
raised
serum
bilirubin, prolonged prothrombin time, and a low serum albumin have a poor prognosis. Treatment with the
A significant correlation was found between serum P3NP and histological stage (p 002; fig 3), the highest levels being found in patients with bridging fibrosis and cirrhosis (Ludwig stages 3 and 4). In addition, a strong correlation was found between serum P3NP and the degree of parenchymal pericellular fibrosis (p < 0-001), although for both indices there was considerable overlap between groups. A positive correlation was seen between serum P3NP and markers of necrosis and inflammation-namely, serum aminotransferases and histological piecemeal necrosis (table IV). Serum P3NP also correlated with cholestasis as assessed by serum bilirubin and alkaline phosphatase concentrations and the degree of copper binding protein deposition in the liver. No correlation was found between serum P3NP and synthetic function as measured by serum albumin and prothrombin time. =
antifibrogenic drug colchicine seemed to improve survival in this group of patients, and further studies on the use of this drug in PBC are essential. Furthermore, our results show that the prognostic value of serum P3NP is independent of other factors shown to affect survival. The prognostic value of histological staging in PBC remains controversial. In a major study from Yale,s cirrhosis was not found to be an independent influence on survival, whilst fibrosis confined to the portal tracts was associated with a good outcome. In contrast, Christensen and colleagues6 showed that the presence of cirrhosis was independently associated with a poor prognosis. In the present study, the presence or absence of cirrhosis was not found, in either the univariate or multivariate analysis, to influence survival. Our data support the suggestion that conventional histological staging of the disease is of limited prognostic value2526 and an inappropriate indicator of response to drug treatment. The high P3NP concentrations in fibrotic liver disease may reflect several different processes.2728 In a rat model of cirrhosis, a raised P3NP predicts the deposition of newly synthesised collagen,29 while in man P3NP has been proposed as a serum marker of hepatic fibrosis8 9,12,30--32 and fibrogenesis. In addition, serum P3NP has been shown to correlate with markers of hepatic inflammation and necrosis34,35 and may also be influenced by cholestasis.36 Fibrosis and fibrogenesis, necrosis and inflammation, and cholestasis are important features of PBC and we have TABLE IV-CORRELATION OF SERUM
P3NP LEVELS WITH DISEASE
INDICES
Fig 3-Serum P3NP levels in PBC patients according to histological stage (Ludwig).
Upper means.
limit of normal is shown
as a
dotted line. Solid lines show group
All p values
are
two-tailed. Correlations with
a
p value of
> 0 05,
are
expressed as NS. AST, ALT = aspartate, alanme aminotransferase. *Only 20% (1050) of biopsy specimens showed histological evidence of cholestasis.
1024
demonstrated correlations between serum P3NP and markers of all these processes. Significantly raised levels were found in Ludwig stage 1, which is characterised by destruction of bile ducts, bile leakage, and chronic inflammatory cell infiltration of portal tracts, all of which may stimulate fibrogenesis.2025.37 From stage II onwards, there is piecemeal necrosis of periportal hepatocytes which, in our experience, is usually associated with pericellular fibrosis38 and which may further stimulate fibrogenesis. The correlations found in the present study between serum P3NP, piecemeal necrosis, and serum transaminase levels support this finding and may also reflect the release of P3NP previously laid down in type III collagen fibrils .17,39 Finally, the association of serum P3NP with bilirubin suggests that in the late stages of the disease, when there is deepening jaundice, impaired biliary excretion of P3NP may become an important factor. It is clear that the concentration of P3NP in serum is an important, independent, prognostic indicator in PBC. Although the factors leading to increased serum concentrations are complex, our data show that the major pathophysiological processes of the disease all contribute, and that the test is therefore a marker of overall disease activity. P3NP should become a valuable clinical tool to monitor response to drug treatment and to predict the need for, and timing of, liver transplantation. We thank the
physicians
and surgeons of the North West who referred
patients to us. Correspondence should be addressed to T. W. W., Liver Unit, University Department of Gastroenterology, Manchester Royal Infirmary, 1Blanchester M13 9WL. REFERENCES 1. Warnes TW. Treatment of primary biliary cirrhosis. Semin Liver Dis 1985; 5: 228-40. 2 Neuberger J. Liver transplantation indications and timing. Curr Opin Gastroenterol 1987, 3: 402-07. 3. Shapiro JM, Smith H, Schaffner F. Serum bilirubin: a prognostic factor in primary biliary cirrhosis. Gut 1979; 20: 137-40. 4. Epstein O, Fraga EG, Keynan A, Sherlock S. When should patients with primary biliary cirrhosis be referred for liver transplantation? Liver 1984; 4: 82 (abstr) 5. Roll J, Boyer JL, Barry D, Klatskin G. The prognostic importance of clinical and histologic features in asymptomatic and symptomatic primary biliary cirrhosis. N Engl J Med 1983; 308: 1-7 6. Christensen E, Neuberger J, Crowe J, et al Beneficial effect of azathioprine and prediction of prognosis in primary biliary cirrhosis. Final results of an international trial Gastroenterology 1985; 89: 1084-91. 7 Epstein O Primary biliary cirrhosis. In: Elias E, Murphy G, eds Bile in health and disease. Proceeding of the 6th BSG-SK&F International Workshop 1985. Published by Smith, Kline & French Laboratories, 1986: 57-61. 8. Rohde H, Vargas L, Hahn E, Kalbfleisch H, Bruguera M, Timpl R. Radioimmunoassay for type III procollagen peptide and its application to human liver disease Eur J Clin Invest 1979; 9: 451-59. 9. Frei A, Zimmermann A, Weigand K. The N-terminal propeptide of collagen type III in serum reflects activity and degree of fibrosis in patients with chronic liver disease. Hepatology 1984, 4: 830-34. 10. Weigand K, Zaugg PY, Frei A, Zimmermann A Long-term follow-up of serum N-terminal propeptide of collagen type III levels in patients with chronic liver disease. Hepatology 1984, 4: 835-38. 1 1. Niemala O, Risteli 1, Sotaniemi EA, Risteli J. Amino-terminal propeptide of type III procollagen in serum in alcoholic liver disease. Gastroenterology 1983; 85: 254-59 12. Galambos MR, Collins DC, Galambos JT A radioimmunoassay procedure for type III procollagen its use in the detection of hepatic fibrosis. Hepatology 1985, 5: 38-42. 13 Bentsen KD, Horslev-Petersen K, Junker P, Juhl
14
15. 16
17.
E, Lorenzen I, and the Copenhagen Hepatitis Acuta Programme Serum aminoterminal procollagen type III in acute viral hepatitis A long term follow-up study Liver 1987; 7: 96-105. McCullough AJ, Stassen WN, Wiesner RH, Czaja AJ Serum type III procollagen peptide concentrations in severe chronic active hepatitis: relationship to cirrhosis and disease activity Hepatology 1987; 7: 49-54. Eriksson S, Zettervall O The N-terminal propeptide of collagen type II I in serum as a prognostic indicator in primary biliary cirrhosis J Hepatol 1986, 2: 370-78. Savolainen E-R, Miettinen TA, Pikkarainen P, Salaspuro MP, Kivirikko KI Enzymes of collagen synthesis and type III procollagen aminopropeptide in the evaluation of d-penicillamine and medroxyprogesterone treatments of primary
biliary cirrhosis Gut 1983, 24: 136-42. Davis BH, Madri JA. An immunohistochemical and serum ELISA study of type I and III procollagen aminopropeptides in primary biliary cirrhosis Am J Pathol 1987, 128: 265-75.
Methods and Devices SUCTION CHAMBER PROTECTION FOR ENDOSCOPE BIOPSY-CHANNEL VALVES
J.
D. HARRISON
D. L. MORRIS
Department of Surgery, Queen’s Medical Centre, Nottingham NG7 2UH THE spread of AIDS by a contaminated endoscope has not been reported, but endoscopists and their assistants could be at risk of infection with HIV or other pathogens by emission of fluid (blood, gastric juice, or saliva) during insertion or removal of biopsy forceps. We describe a negative pressure chamber biopsy-channel valve, designed by one of us (D. L. M.) to reduce this emission of fluid.
Device A plastic negative pressure chamber with rubber slit valves at each end (figure) replaces the standard endoscope biopsy-channel valve; negative pressure is maintained by connection to a standard suction apparatus. The valve is designed to prevent leakage of fluid around the biopsy forceps caused by positive pressure within the
C BABBS AND OTHERS:
REFERENCES—continued
18. Wames TW, Smith A, Lee FI, Haboubi NY, Johnson PJ, Hunt L A controlled tnal of colchicine in primary biliary cirrhosis. Trial design and preliminary report.J Hepatol 1987; 5: 1-7. 19. Sherlock S, Scheuer PJ The presentation and diagnosis of 100 patients with primary biliary cirrhosis N Engl J Med 1973; 289: 674-78. 20. Ludwig J, Dickson ER, McDonald GSA. Staging of chronic non-suppurative destructive cholangitis (syndrome of primary biliary cirrhosis). Virchows Arch (A )
1978; 379: 103-12 21. Sweat F, Puchtler H, Rosenthal SI Sirius red Fab as a
stain for connective tissue. Arch Pathol 1964, 78: 69-72. 22 Peto R, Pike MC, Armitage P, et al. Design and analysis of randomised clinical trials requiring prolonged observation of each patient Br J Cancer 1977; 35: 1-39. 23. Cox DR. Regression models and life tables. J R Statist Soc, ser B 1972; 34: 187-202 24. Neuberger J, Altman D, Chnstensen E, Tygstrup N, Williams R. Use of a prognostic index m evaluation of liver transplantation for primary biliary cirrhosis Transplantation 1986; 4: 713-16. 25. MacSween RNM, Sumithran E. Histopathology of primary biliary cirrhosis Semin Liver Dis 1981; 1: 282-92. 26. Scheuer PJ Liver biopsy interpretation, 4th ed London: Baillière-Tindall, 1987. 27. Rojkind M The blue glass and the predictive value of serum amino-terminal propeptide of type III procollagen as a marker of liver fibrosis. Hepatology 1984, 4: 977-78. 28. Alcorn J, Chojkier M. Procollagen III peptide (PIIIP): can it reflect hepatic fibrosis? Hepatology 1987, 7: 981-83. 29. Schuppan D, Dumont JM, Kim KY, Hennings G, Hahn EG Serum concentration of the aminoterminal procollagen type III peptide in the rat reflects early formation of connective tissue in experimental liver cirrhosis. J Hepatol 1986, 3: 27-37. 30. Tanaka Y, Mmato Y, Hasamura Y, Takeuchi J Evaluation of hepatic fibrosis by serum proline and amino-terminal type III procollagen peptide levels in alcoholic patients. Dig Dis Sci 1986; 31: 712-17. 31. Sato S, Nouchi T, Womer TM, Lieber CS. Liver fibrosis in alcoholics. Detection by Fab radioimmunoassay of serum procollagen III peptides JAMA 1986, 256: 1471-73. 32. Hahn EG. Blood analysis for liver fibrosis. J Hepatol 1984, 1: 67-73 33. Torres-Salinas MM, Pares A, Caballeria J, et al. Serum procollagen type III peptide as a marker of hepatic fibrogenesis m alcoholic hepatitis. Gastroenterology 1986, 90: 1241-46 34. Surrenti C, Casini A, Milani S, Ambu S, Ceccatelli P, D’Agata A. Is determination of serum N-terminal procollagen type III peptide (sPIIIP) a marker of hepatic fibrosis? Dig Dis Sci 1987, 32: 705-09. 35. Salvolainen ER, Goldberg B, Leo MA, Velez M, Lieber CS. Diagnostic value of serum procollagen peptide measurements in alcoholic liver disease. Alcoholism Clin Exp Res Clin Exp Res 1984; 8: 384-89. 36. Raedsch R, Stiehl A, Sieg A, Walker S, Kommerell B Biliary excretion of procollagen type III peptide in healthy humans and in patients with alcoholic cirrhosis of the liver. Gastroenterology 1983; 85: 1265-70. 37. Sever JM Mediators of increased collagen synthesis in fibrosing organs. Fund Appl Toxicol 1985, 5: 228-39. 38. Haboubi NY, Babbs C, Dawson DJ, Smith A, Wames TW. Nodular hyperplasia of the liver in primary biliary cirrhosis of early histological stages. Am J Gastroenterology 1987; 82: 1100 39. Davis BH, Madri JA. Type I and type III procollagen peptides during hepatic fibrogenesis. An immunohistochemical and ELISA serum study in the CCl4 rat model. Am J Pathol 1987; 126: 137-47
should allow logical treatment for the disabling postural hypotension that usually accompanies autonomic failure. For example, in patients with "flat" noradrenaline responses to edrophonium it would be inappropriate to use a drug that stimulates noradrenaline release.
Furthermore, results of such
a test
We thank Dr R. J. Prescott, Medical Computing and Statistics Unit, University of Edinburgh for his help with the statistical analysis; Dr R. A. Riemersma, Cardiovascular Research Unit, University of Edinburgh, for his help with the measurements of plasma noradrenaline; and Dr P. Matute for his assistance with the studies themselves. The study was supported by a
grant from the Wellcome Trust.
Correspondence should be addressed to D. J. E., Department of Medicine, Royal Infirmary, Edinburgh EH3 9YW. REFERENCES 1 Bannister R. Introduction and classification In Bannister R, ed Autonomic failure. Oxford- Oxford University Press, 1983 1-13
TYPE III PROCOLLAGEN PEPTIDE: A MARKER OF DISEASE ACTIVITY AND PROGNOSIS IN PRIMARY BILIARY CIRRHOSIS
CHRISTOPHER BABBS1 LINDA P. HUNT2 NAJIB Y. HABOUBI3
ALEXANDER SMITH1 BRENDA P. ROWAN1 THOMAS W. WARNES1
Liver Unit, University Department of Gastroenterology,1 and Faculty of Medicine Computational Group,2 Manchester Royal Infirmary; and Department of Pathology, University Hospital of South Manchester3
The prognostic value of the aminoterminal propeptide of type III procollagen (P3NP) was investigated in 63 patients with primary biliary cirrhosis (PBC) followed for up to 87 months. No patient with an initially normal serum P3NP level died during the study; survival was significantly worse with increasing serum P3NP levels. Cox multivariate analysis confirmed that serum P3NP was an independent prognostic variable. Positive correlations were found between serum P3NP and histological stage, pericellular fibrosis, piecemeal necrosis, and serum concentrations of alanine aminotransferase and aspartate aminotransferase. Raised P3NP levels also correlated with the degree of cholestasis as evaluated by serum bilirubin, serum alkaline phosphatase, and copper binding protein deposition in the liver. Serum P3NP is of prognostic value because it reflects the major pathophysiological features of PBC.
Summary
JG, Tuck RR. Disorders of the autonomic nervous system. part 1. Pathophysiology and clinical features. Ann Neurol 1987, 21: 419-30. 3 Oppenheimer D Neuropathology of progressive autonomic failure. In. Bannister R, ed Autonomic failure. Oxford: Oxford University Press, 1983; 267-83. 4. Leveston SA, Shah SD, Cryer PE Cholinergic stimulation of norepinephrine release in man. Evidence of a sympathetic postganglionic axonal lesion in diabetic adrenergic neuropathy. J Clin Invest 1979, 64: 374-80 5. Ewing DJ, Bellavere F, Espi F, et al. Correlation of cardiovascular and neuroendocrine tests of autonomic function in diabetes. Metabolism 1986, 35: 349-53. 6. Matthews DM, Martyn CN, Riemersma RA, Clarke BF, Ewing DJ. Noradrenaline response to edrophonium (Tensilon) and its relation to other autonomic tests in diabetic subjects Diabetes Res 1987, 6: 175-80 7. Ewing DJ, Martyn CN, Young RJ, Clarke BF The value of cardiovascular autonomic function tests 10 years’ experience in diabetes Diabetes Care 1985; 8: 491-98. 8. Da Prada M, Zurcher G Simultaneous radioenzymatic determination of plasma and tissue adrenaline, noradrenaline and dopamine within the femtomole range. Life Sci 1976, 19: 1161-73. 9. Taylor P. Anticholinesterase agents In Gilman AG, Goodman LS, Rall TW, Murad F, eds Goodman and Gilman’s pharmacological basis of therapeutics, 7th ed New York Macmillan, 1985 110-29 10. Sever PS Plasma noradrenaline in autonomic failure. In: Bannister R, ed. Autonomic failure Oxford: Oxford University Press, 1983; 155-73 11. Polinsky RJ. Pharmacological responses and biochemical changes in progressive autonomic failure. In. Bannister R, ed. Autonomic failure. Oxford: Oxford University Press, 1983:201-36. 2 McLeod
the diagnosis and follow-up of patients with acute and chronic liver disease9-13 and in the assessment of disease activity in autoimmune chronic active hepatitis.14 The interpretation of raised serum P3NP concentrations in PBC has been complicated by the small number of patients studied;9,15-17 one report suggests that the test may be of prognostic value.155 The aim of the present study was to examine the prognostic value of serum P3NP in a large group of PBC patients and to compare it with other factors previously shown to influence survival. In addition, we wished to test our hypothesis that this assay might reflect overall disease activity by relating serum levels to the histological stage of the disease and the degree of fibrosis, as well as to markers of necrosis, inflammation, and cholestasis.
Patients and Methods The study group consisted of 63 patients (57 female; mean age 57-8 years, range 29-78) prospectively evaluated in our unit before entry into a controlled trial of colchicine therapy’8 and followed for a mean of 37 months (range 0 6—87). PBC was diagnosed by conventional criteria’9 and patients were divided into those with
typical symptoms (pruritus, pigmentation, jaundice, ascites, or gastrointestinal bleeding; n = 28) and those who were presymptomatic (n — 35). The colchicine and placebo groups were well matched at entry for all measured indices (table I). There were 22 deaths during the study and in 19 cases this was the result of liver disease. 3 patients died of unrelated causes (1 carcinoma rectum; 1 aplastic anaemia; 1 pneumonia) and these cases were censored from the survival analysis.
Introduction WITH the advent of liver transplantation as an established treatment for patients with end-stage primary biliary cirrhosis (PBC), the assessment of prognosis has become of great clinical importance.’Serum bilirubin has consistently been shown to be an important prognostic guide in PBC;1,3-6 however, the disease is clinically heterogeneous and assessment of prognosis cannot be based on measurement of a single variable.7 The prognostic value of other clinical, biochemical, and histological indices remains controversial. The aminoterminal propeptide of type III procollagen (P3NP) is released during the extracellular processing of type III collagen, a major component of the hepatic extracellular matrix, and may be measured in serum by radioimmunoassay.8 The test has proved of clinical value in
TABLE I-GROUP COMPARABILITY
*Geometnc mean (range) for tArithmetric mean (SD).
log transformed
data.
1022 TABLE II—UNIVARIATE ANALYSIS
Results The upper limit of normal for serum P3NP (mean + 2 SD) was 13 8 ng/ml and concentrations exceeded this in 73% (46/63) of PBC patients (p < 0 001, fig 1). The effect on survival, of the serum P3NP level at entry to the study, is shown in fig 2. No deaths from liver disease were recorded among the 17 patients with initially normal serum P3NP values (group A) although 9 of these patients had bridging fibrosis or cirrhosis (Ludwig stages 3 and 4, fig 3) and 5 had a raised serum bilirubin. The difference in cumulative survival between the three groups was highly significant
(p < 0001).
Effect of different variables survival curves (logrank test).
on
survival estimated from
Kaplan-Meier
Serum was separated soon after collection and stored at - 20°C until assay. P3NP levels were measured by radioimmunoassay (Behringwerke Hoechst, Frankfurt) as described by Rohde et al. 8 A normal reference range was established in our laboratory in 65 healthy adults. The intra-assay coefficient of variation (CV) was 85 % and the inter-assay CV, 12 % (n 7). Liver biopsy specimens were fixed in formalin and staged according to the criteria of Ludwig et al ;20 10 patients were stage 1, 11stage 2, 17 stage 3, and 25 stage 4 (established cirrhosis). The following histological indices were assessed blindly and graded semiquantitatively 0 to + 3: piecemeal necrosis, parenchymal inflammation, cholestasis, and copper binding protein deposition. Parenchymal pericellular (perisinusoidal) fibrosis was also graded 0 to + 3 after staining with haematoxylin-picrosirius red.21 Conventional liver function tests were done by standard =
autoanalyser techniques. Correlation coefficients were calculated by means of Pearson and Kendall non-parametric tests and two-tailed levels of significance are provided throughout. Logarithmic conversion was used to normalise skewed distribution of serum bilirubin, alkaline phosphatase, and prothrombin index. To estimate the relation between each of the 17 variables assessed and survival we applied the logrank test22 to Kaplan-Meier survival curves after dividing the patients into appropriate subgroups (table II). Those variables with a significant influence on prognosis and those that showed a trend towards significance were entered into a Cox multivariate analysis, the proportional hazards model being used to establish independent significance.23 TABLE III-FINAL COX MODEL
*Exponential transformation of age was performed as previously suggested6 positive value for the regression coefficient indicates an adverse influence on survival and vice versa for a negative value. A coefficient of 0 would imply
A
no
effect
on
survival.
Fig
1—Serum P3NP levels in
a
normal group and PBC
Histograms show the means with standard deviation standard deviations (bars).
In addition to indicated that log
serum
serum
patients.
(solid blocks)
and 2
P3NP levels, univariate analysis bilirubin, log prothrombin index,
symptom status, and serum aspartate aminotransferase all had a significant influence on survival (table II). Age, serum albumin, presence or absence of cirrhosis, and treatment status all showed a trend towards prognostic importance but did not reach statistical significance. These factors were, however, included in the Cox multivariate analysis since variables showing a trend toward significance in a univariate analysis may become significant when other factors are adjusted for. The results of this analysis (table III) confirm the previously recognised prognostic value of serum bilirubin, serum albumin, prothrombin index, age, and symptom status. Serum P3NP remains a highly significant prognostic variable which is independent of the other variables in the model. In contrast, the presence or absence of cirrhosis had no independent influence on survival; treatment with colchicine seemed to improve survival, when other factors were adjusted for.
histological cholestasis,
1023
Discussion The present study clearly demonstrates that serum P3NP is an important predictor of survival in PBC and suggests that prognosis is related to the degree to which P3NP is raised. Thus, none of the patients whose initial serum P3NP level was normal (group A) died, despite the fact that 9 of this group had histologically late stage disease. Outcome was worst in those patients whose initial serum P3NP was above the mean value (group C): 52 % (15/29) of these patients died
Fig 2-Cumulative proportion of patients surviving initial P3NP levels (Kaplan-Meier). < 13-8 ng/ml 19 ng/ml (n = 29).
Group A, C
>
(n
=
17); group B,
13 9-19-9
at
ng/ml (n
=
different
17); group
during follow-up. In previous reports there is lack of agreement on the prognostic value of factors other than serum bilirubin 4-6 In the present study, using the Cox proportional hazards model, we have shown that older patients, those presenting with symptoms, and those who have
a
raised
serum
bilirubin, prolonged prothrombin time, and a low serum albumin have a poor prognosis. Treatment with the
A significant correlation was found between serum P3NP and histological stage (p 002; fig 3), the highest levels being found in patients with bridging fibrosis and cirrhosis (Ludwig stages 3 and 4). In addition, a strong correlation was found between serum P3NP and the degree of parenchymal pericellular fibrosis (p < 0-001), although for both indices there was considerable overlap between groups. A positive correlation was seen between serum P3NP and markers of necrosis and inflammation-namely, serum aminotransferases and histological piecemeal necrosis (table IV). Serum P3NP also correlated with cholestasis as assessed by serum bilirubin and alkaline phosphatase concentrations and the degree of copper binding protein deposition in the liver. No correlation was found between serum P3NP and synthetic function as measured by serum albumin and prothrombin time. =
antifibrogenic drug colchicine seemed to improve survival in this group of patients, and further studies on the use of this drug in PBC are essential. Furthermore, our results show that the prognostic value of serum P3NP is independent of other factors shown to affect survival. The prognostic value of histological staging in PBC remains controversial. In a major study from Yale,s cirrhosis was not found to be an independent influence on survival, whilst fibrosis confined to the portal tracts was associated with a good outcome. In contrast, Christensen and colleagues6 showed that the presence of cirrhosis was independently associated with a poor prognosis. In the present study, the presence or absence of cirrhosis was not found, in either the univariate or multivariate analysis, to influence survival. Our data support the suggestion that conventional histological staging of the disease is of limited prognostic value2526 and an inappropriate indicator of response to drug treatment. The high P3NP concentrations in fibrotic liver disease may reflect several different processes.2728 In a rat model of cirrhosis, a raised P3NP predicts the deposition of newly synthesised collagen,29 while in man P3NP has been proposed as a serum marker of hepatic fibrosis8 9,12,30--32 and fibrogenesis. In addition, serum P3NP has been shown to correlate with markers of hepatic inflammation and necrosis34,35 and may also be influenced by cholestasis.36 Fibrosis and fibrogenesis, necrosis and inflammation, and cholestasis are important features of PBC and we have TABLE IV-CORRELATION OF SERUM
P3NP LEVELS WITH DISEASE
INDICES
Fig 3-Serum P3NP levels in PBC patients according to histological stage (Ludwig).
Upper means.
limit of normal is shown
as a
dotted line. Solid lines show group
All p values
are
two-tailed. Correlations with
a
p value of
> 0 05,
are
expressed as NS. AST, ALT = aspartate, alanme aminotransferase. *Only 20% (1050) of biopsy specimens showed histological evidence of cholestasis.
1024
demonstrated correlations between serum P3NP and markers of all these processes. Significantly raised levels were found in Ludwig stage 1, which is characterised by destruction of bile ducts, bile leakage, and chronic inflammatory cell infiltration of portal tracts, all of which may stimulate fibrogenesis.2025.37 From stage II onwards, there is piecemeal necrosis of periportal hepatocytes which, in our experience, is usually associated with pericellular fibrosis38 and which may further stimulate fibrogenesis. The correlations found in the present study between serum P3NP, piecemeal necrosis, and serum transaminase levels support this finding and may also reflect the release of P3NP previously laid down in type III collagen fibrils .17,39 Finally, the association of serum P3NP with bilirubin suggests that in the late stages of the disease, when there is deepening jaundice, impaired biliary excretion of P3NP may become an important factor. It is clear that the concentration of P3NP in serum is an important, independent, prognostic indicator in PBC. Although the factors leading to increased serum concentrations are complex, our data show that the major pathophysiological processes of the disease all contribute, and that the test is therefore a marker of overall disease activity. P3NP should become a valuable clinical tool to monitor response to drug treatment and to predict the need for, and timing of, liver transplantation. We thank the
physicians
and surgeons of the North West who referred
patients to us. Correspondence should be addressed to T. W. W., Liver Unit, University Department of Gastroenterology, Manchester Royal Infirmary, 1Blanchester M13 9WL. REFERENCES 1. Warnes TW. Treatment of primary biliary cirrhosis. Semin Liver Dis 1985; 5: 228-40. 2 Neuberger J. Liver transplantation indications and timing. Curr Opin Gastroenterol 1987, 3: 402-07. 3. Shapiro JM, Smith H, Schaffner F. Serum bilirubin: a prognostic factor in primary biliary cirrhosis. Gut 1979; 20: 137-40. 4. Epstein O, Fraga EG, Keynan A, Sherlock S. When should patients with primary biliary cirrhosis be referred for liver transplantation? Liver 1984; 4: 82 (abstr) 5. Roll J, Boyer JL, Barry D, Klatskin G. The prognostic importance of clinical and histologic features in asymptomatic and symptomatic primary biliary cirrhosis. N Engl J Med 1983; 308: 1-7 6. Christensen E, Neuberger J, Crowe J, et al Beneficial effect of azathioprine and prediction of prognosis in primary biliary cirrhosis. Final results of an international trial Gastroenterology 1985; 89: 1084-91. 7 Epstein O Primary biliary cirrhosis. In: Elias E, Murphy G, eds Bile in health and disease. Proceeding of the 6th BSG-SK&F International Workshop 1985. Published by Smith, Kline & French Laboratories, 1986: 57-61. 8. Rohde H, Vargas L, Hahn E, Kalbfleisch H, Bruguera M, Timpl R. Radioimmunoassay for type III procollagen peptide and its application to human liver disease Eur J Clin Invest 1979; 9: 451-59. 9. Frei A, Zimmermann A, Weigand K. The N-terminal propeptide of collagen type III in serum reflects activity and degree of fibrosis in patients with chronic liver disease. Hepatology 1984, 4: 830-34. 10. Weigand K, Zaugg PY, Frei A, Zimmermann A Long-term follow-up of serum N-terminal propeptide of collagen type III levels in patients with chronic liver disease. Hepatology 1984, 4: 835-38. 1 1. Niemala O, Risteli 1, Sotaniemi EA, Risteli J. Amino-terminal propeptide of type III procollagen in serum in alcoholic liver disease. Gastroenterology 1983; 85: 254-59 12. Galambos MR, Collins DC, Galambos JT A radioimmunoassay procedure for type III procollagen its use in the detection of hepatic fibrosis. Hepatology 1985, 5: 38-42. 13 Bentsen KD, Horslev-Petersen K, Junker P, Juhl
14
15. 16
17.
E, Lorenzen I, and the Copenhagen Hepatitis Acuta Programme Serum aminoterminal procollagen type III in acute viral hepatitis A long term follow-up study Liver 1987; 7: 96-105. McCullough AJ, Stassen WN, Wiesner RH, Czaja AJ Serum type III procollagen peptide concentrations in severe chronic active hepatitis: relationship to cirrhosis and disease activity Hepatology 1987; 7: 49-54. Eriksson S, Zettervall O The N-terminal propeptide of collagen type II I in serum as a prognostic indicator in primary biliary cirrhosis J Hepatol 1986, 2: 370-78. Savolainen E-R, Miettinen TA, Pikkarainen P, Salaspuro MP, Kivirikko KI Enzymes of collagen synthesis and type III procollagen aminopropeptide in the evaluation of d-penicillamine and medroxyprogesterone treatments of primary
biliary cirrhosis Gut 1983, 24: 136-42. Davis BH, Madri JA. An immunohistochemical and serum ELISA study of type I and III procollagen aminopropeptides in primary biliary cirrhosis Am J Pathol 1987, 128: 265-75.
Methods and Devices SUCTION CHAMBER PROTECTION FOR ENDOSCOPE BIOPSY-CHANNEL VALVES
J.
D. HARRISON
D. L. MORRIS
Department of Surgery, Queen’s Medical Centre, Nottingham NG7 2UH THE spread of AIDS by a contaminated endoscope has not been reported, but endoscopists and their assistants could be at risk of infection with HIV or other pathogens by emission of fluid (blood, gastric juice, or saliva) during insertion or removal of biopsy forceps. We describe a negative pressure chamber biopsy-channel valve, designed by one of us (D. L. M.) to reduce this emission of fluid.
Device A plastic negative pressure chamber with rubber slit valves at each end (figure) replaces the standard endoscope biopsy-channel valve; negative pressure is maintained by connection to a standard suction apparatus. The valve is designed to prevent leakage of fluid around the biopsy forceps caused by positive pressure within the
C BABBS AND OTHERS:
REFERENCES—continued
18. Wames TW, Smith A, Lee FI, Haboubi NY, Johnson PJ, Hunt L A controlled tnal of colchicine in primary biliary cirrhosis. Trial design and preliminary report.J Hepatol 1987; 5: 1-7. 19. Sherlock S, Scheuer PJ The presentation and diagnosis of 100 patients with primary biliary cirrhosis N Engl J Med 1973; 289: 674-78. 20. Ludwig J, Dickson ER, McDonald GSA. Staging of chronic non-suppurative destructive cholangitis (syndrome of primary biliary cirrhosis). Virchows Arch (A )
1978; 379: 103-12 21. Sweat F, Puchtler H, Rosenthal SI Sirius red Fab as a
stain for connective tissue. Arch Pathol 1964, 78: 69-72. 22 Peto R, Pike MC, Armitage P, et al. Design and analysis of randomised clinical trials requiring prolonged observation of each patient Br J Cancer 1977; 35: 1-39. 23. Cox DR. Regression models and life tables. J R Statist Soc, ser B 1972; 34: 187-202 24. Neuberger J, Altman D, Chnstensen E, Tygstrup N, Williams R. Use of a prognostic index m evaluation of liver transplantation for primary biliary cirrhosis Transplantation 1986; 4: 713-16. 25. MacSween RNM, Sumithran E. Histopathology of primary biliary cirrhosis Semin Liver Dis 1981; 1: 282-92. 26. Scheuer PJ Liver biopsy interpretation, 4th ed London: Baillière-Tindall, 1987. 27. Rojkind M The blue glass and the predictive value of serum amino-terminal propeptide of type III procollagen as a marker of liver fibrosis. Hepatology 1984, 4: 977-78. 28. Alcorn J, Chojkier M. Procollagen III peptide (PIIIP): can it reflect hepatic fibrosis? Hepatology 1987, 7: 981-83. 29. Schuppan D, Dumont JM, Kim KY, Hennings G, Hahn EG Serum concentration of the aminoterminal procollagen type III peptide in the rat reflects early formation of connective tissue in experimental liver cirrhosis. J Hepatol 1986, 3: 27-37. 30. Tanaka Y, Mmato Y, Hasamura Y, Takeuchi J Evaluation of hepatic fibrosis by serum proline and amino-terminal type III procollagen peptide levels in alcoholic patients. Dig Dis Sci 1986; 31: 712-17. 31. Sato S, Nouchi T, Womer TM, Lieber CS. Liver fibrosis in alcoholics. Detection by Fab radioimmunoassay of serum procollagen III peptides JAMA 1986, 256: 1471-73. 32. Hahn EG. Blood analysis for liver fibrosis. J Hepatol 1984, 1: 67-73 33. Torres-Salinas MM, Pares A, Caballeria J, et al. Serum procollagen type III peptide as a marker of hepatic fibrogenesis m alcoholic hepatitis. Gastroenterology 1986, 90: 1241-46 34. Surrenti C, Casini A, Milani S, Ambu S, Ceccatelli P, D’Agata A. Is determination of serum N-terminal procollagen type III peptide (sPIIIP) a marker of hepatic fibrosis? Dig Dis Sci 1987, 32: 705-09. 35. Salvolainen ER, Goldberg B, Leo MA, Velez M, Lieber CS. Diagnostic value of serum procollagen peptide measurements in alcoholic liver disease. Alcoholism Clin Exp Res Clin Exp Res 1984; 8: 384-89. 36. Raedsch R, Stiehl A, Sieg A, Walker S, Kommerell B Biliary excretion of procollagen type III peptide in healthy humans and in patients with alcoholic cirrhosis of the liver. Gastroenterology 1983; 85: 1265-70. 37. Sever JM Mediators of increased collagen synthesis in fibrosing organs. Fund Appl Toxicol 1985, 5: 228-39. 38. Haboubi NY, Babbs C, Dawson DJ, Smith A, Wames TW. Nodular hyperplasia of the liver in primary biliary cirrhosis of early histological stages. Am J Gastroenterology 1987; 82: 1100 39. Davis BH, Madri JA. Type I and type III procollagen peptides during hepatic fibrogenesis. An immunohistochemical and ELISA serum study in the CCl4 rat model. Am J Pathol 1987; 126: 137-47