Intra- and extracellular'alpha,-antitrypsin in liver with

J Clin Pathol 1981;34:1020-1025

Intra- and extracellular'alpha,-antitrypsin in liverdisease with special reference to Pi phenotypeJOYCE CARLSON, STEN ERIKSSON, INGA HXGERSTRAND

From the Department of Medicine, University of Lund, Malmo General Hospital, Malmo, Swedenand the Institute ofPathology, University ofOdense, Odense, Denmark

SUMMARY In order to study the relation between intra- and extrahepatocellular alpha1-antitrypsin(a,-AT) concentrations in patients with various Pi phenotypes, a prospective series of needle liverbiopsies was stained with both periodic acid-Schiff (PAS) and a specific immunoperoxidase techniqueto demonstrate intracellular ocx-AT. Concomitant blood samples from all patients were analysed foro1-AT. Pi phenotypes were determined by isoelectric focusing.

Non-globular intrahepatocellular oc1-AT can be seen in biopsies from Pi M patients with increasedplasma a1-AT concentrations and active liver disease. No evidence was found in this study of 250patients (including 22 controls) for predisposition toward liver disease in any phenotypic group. PASor immunoperoxidase staining (or both) for ac,-AT demonstrated characteristic globular inclusionsin 11 of 15 cases having the Z allele, one case being diffusely positive and three negative. Biopsies from3 of 207 patients with liver disease and lacking the Z allele had globular inclusions seen with both PASand immunoperoxidase techniques. a,-AT globules in absence of the Z allele are most often found inelderly patients with severe disease and high plasma al-AT concentrations.

Alphal-antitrypsin (ocm-AT, mi-protease inhibitor) isan acute phase reactant plasma protein with broadspectrum antiprotease activity synthesised by thehepatocytes. A selective increase of its plasmaconcentration is often seen in liver disease and thislevel generally seems to reflect the activity of thedisease.' Low plasma concentrations are seen insome phenotypes. Ofspecial interest is the PiZpheno-type in which increased frequencies of fibrosis, cir-rhosis and hepatoma23 have been reported. Neonatalhepatitis occurs in 10% of homozygous Pi Z childrenand increased hepatic enzyme activity is found inapproximately 50% of Pi children.4 Typical PAS-positive inclusion bodies, first described by Sharpet al.,5 and consisting of aggregated oti-AT lackingsialic acid and with an amino acid substitution6 areseen in Pi Z hepatocytes. Such inclusions were firstthought to be pathognomonic for the Z allele7 butseveral reports during the past few years have dis-puted this assumption. Heterozygous Pi MZ individ-uals also have globular inclusions and one case ofhepatocellular carcinoma in a 16-year-old MZ boyhas been reported,8 but most investigators havefound no evidence ofincreased liver disease frequencyin such individuals.

Accepted for publication 7 January 1981

xcx-AT may have an important protective role asa protease inhibitor in liver disease. The mechanismsregulating the plasma concentration in both normaland other phenotypes are, however, incompletelyknown. To gain insight into these mechanisms wehave studied (i) the relation between intrahepato-cellular immunoreactive oc1-AT and plasma concen-trations in various liver diseases; (ii) the frequencyand some clinical conditions associated with globularinclusions (ml-AT globules) in absence of the Zallele and (iii) the distribution of phenotypic groupsamong patients with liver disease. The study wasperformed prospectively using an oci-AT-specificimmunoperoxidase staining in addition to routinePAS-staining of 250 liver biopsies (including 22controls) for visualisation of intracellular oci-ATand isoelectric focusing for phenotype determinationin all cases.

Patients, material and methods

The material comprises 250 consectutive needle liverbiopsies obtained from patients admitted to MalmoGeneral Hospital. It includes 22 control biopsiesobtained with informed consent from patients withnormal liver function undergoing elective chole-cystectomy, according to the principles of the Ethical

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Intra- and extracellular alphal-antitrypsin in liver disease with special reference to Pi phenotype

Committee of the hospital. The diagnoses were

alcoholic liver disease in 88 cases (cirrhosis in 20),chronic active hepatitis (CAH) 15; chronic persistenthepatitis 5; primary biliary cirrhosis 10; venouscongestion 6; toxic liver disease 6; hepatoma 6; viralhepatitis 4; systemic disease with liver involvementin 52 cases and diverse or complex diagnoses in theremaining cases. Only one biopsy from each patientwas included. Blood samples were obtained from allpatients within 24 h of needle liver biopsy forquantitative xc1-AT and phenotype determinations.Plasma ml-AT concentrations were determined atthe Department of Clinical Chemistry by an electro-immunoassay technique using a mono-specific anti-serum available at the laboratory. Phenotypeswere analysed by isoelectric focusing in polyacryl-amide gels using a modification of the method ofPierce et al.9 We thank Dr Jan-Olof Jeppsson forreviewing phenotype designations. All liver biopsieswere routinely stained with haematoxylin and eosin,Gordon-Sweet's reticulin, van Gieson's stain andPAS-staining after diastase digestion. Biopsies were

judged using only the internal code from theDepartment of Pathology for identification. Scoreswere given for the pathological parameters: bridgingnecrosis, piece-meal necrosis, fibrosis, cirrhosis,portal inflammation, centrilobular necrosis, bile-duct replication, Kupffer-cell activity, steatosis,cholestasis and subjective impression of inflamma-tory activity.' 10 Untreated paraffin sections of 242biopsies were examined separately and independentlyusing the indirect peroxidase conjugate method re-

ported by Taylor." Rabbit antiserum, found to bemonospecific for human oxl-AT by crossed immuno-electrophoresis'2 was used in a dilution 1/20 insteadof human immunoglobulin components and car-bazol was substituted for diaminobenzidine.13 Per-oxidase-conjugated swine antirabbitIgG was used in a1/50 dilution. All biopsies which stained positivelywith this technique were also stained with rabbitantisera against albumin, fibrinogen and IgG.Antisera were obtained from DAKO, Copenhagen,Denmark. Results of the immunoperoxidase examin-

ation were compared with the diastase-treated PAS-stained biopsies.

Results

Distribution ofphenotypes andgenefrequencies amongpatients with liver disease compared to Scandi-navian normal populations is shown in Table 1. Aslight but statistically insignificant (p > 04 10)overrepresentation of the Z and S alleles was foundamong patients with liver disease. No significantdifferences were found in mean age, total biopsyscore, inflammatory activity, degree of fibrosis orcirrhosis between phenotypic groups (Table 2). Twoof 11 patients with phenotype MS had chronic activehepatitis (13 % of all CAH patients) whereas only 6%of all patients with liver disease had this diagnosis.Subtypes of the M phenotype could easily be dis-tinguished in 14% of M phenotypes after electro-focusing. No evidence was present for any case ofM Malton orM Duarte in this material.

Immunoperoxidase staining of al-AT was performedon all 242 biopsies with sufficient material forinvestigation (including all controls). Hepatocytesin a minority of biopsies (28/227) from patientslacking the Z allele stained diffusely positive foral,-AT. Mean plasma oti-AT concentrations, biopsyscores and values for inflammatory activity inbiopsies were calculated and compared to those fornegative biopsies. Among Pi M patients in the posi-tive group the mean ml-AT concentration was1-9 ± 0 7 (SD) and 0 2 g/l higher (0 32 > p >

0 10) than for patients with negative biopsies. Meanbiopsy score was 7 6 ± 5-1 (SD) compared to5 * 3 ± 4 * 4 (0 05 > p > 0 * 01). Inflammatoryactivities were I 4 ± 1 * 0 and 1 0 ± 0 - 9 (0* 05 > p> 0.01) for the two groups, respectively. Biopsiesfrom 102 patients showed positive (xi-AT staining inleucocytes, Kupffer's cells or round cells, but nodifferences were found between mean plasma al-ATconcentrations, biopsy scores or activities in theseand patients with negative biopsies.

Table 1 Distribution ofphenotypes andgenefrequencies

Phenotype MM M- MS MZ FM SS SZ ZZ

2830 healthy Norwegians (14) 2540 116 81 72 4 4 2228 biopsied patients with liver disease 199 1 12 11 3 0 1 1

Genefrequencies PiM Pi S Pi Z Pi F Other

2830healthyNorwegians(14) 0*946 0*023 0*0157 0*0133 0 0026995 adult Swedes (15) 0 0240228 patients with liver disease 0 932 0 0285 0 0307 0 0066 0 002

The probability that the frequency of the Z gene in liver patients is the same as that among normal Norwegians and Swedes is 0 * 50 > p > 0 *I0using the x' test. The major contribution to the difference arises in the Z gene group.

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Table 2 Correlation between phenotype, liver biopsy characteristics, age and sex among 228 patients with liver disease

Phenotype n Mean age Y% male Biopsy score Biopsy activity Fibrosis Cirrhosis

Pi M 199 53 ± 14 59 6-1 44 5 11-I 0 9 0 7 00-9(107) 0-4 08-0 (48)Pi M- 1 27 0 0 0 0 (0) 0 (0)Pi FM 3 52 19 33 7-2 ±7-4 1 *2 0-8 1 0 ±1 7(1) 0 7 i 1 1(1)Pi MS 12 51 17 45 6 5 4 -7 1 2 - -2 0 8 0 7 (8) 0 1 --0 3 (2)PiMZ 11 50 15 56 5 9±3-7 1l1 I 0 0 9- 1 0(5) 0 4 1-0(2)Pi SZ 46 100 0 5 0 0 5 (0) 0 (0)Pi ZZ 1 39 100 5 5 1 5 0 (0) 0 (0)

Biopsy score is based on the sum of characteristics listed in the text (see ref. 14). Maximal possible score was 30. Maximal degree of the singlecharacteristics activity, fibrosis and cirrhosis is 3 points each. The number of patients within each phenotypic group having biopsy signs of fibrosisor cirrhosis is shown in parentheses.

No statistically significant differences were found between any of the phenotypic groups.

Fifteen cases were found to have (xi-AT specificglobules in the hepatocytes including 10 cases ofPi MZ (two controls) and one Pi ZZ. Of these 11 cases,

three MZ biopsies were PAS-negative. Three cases

of Pi MZ and 1 Pi SZ were negative to both immuno-peroxidase and PAS-staining. Of the 207 cases studiedwith liver disease and lacking the Z allele, 3 hadimmunoperoxidase and PAS-positive globules in thehepatocytes. Their clinical data, phenotypes, biopsyscores and plasma aci-AT concentrations are

summarised in Table 3 together with similar cases

from published reports. Globules in our patientwith adenocarcinoma had typical appearance foroxi-AT globules and were present in approximately10 normal hepatocytes adjacent to metastatic tissue.In the case with venous congestion 50-100 hepato-cytes with immunoreactive txl-AT of both diffuse andglobular appearance were found periportally as wellas centrilobularly in one lobulus (Fig. 1). In thiscase alone staining was also positive for IgG,

fibrinogen and albumin. The third case (rheuma-toid arthritis) had globules with appearance andlocation typical for those seen in Pi Z deficiency(Fig. 2).

Discussion

Opinions on the occurrence of liver disease among

MZ individuals are divided. Eriksson et al.9 reportedan increased incidence of cirrhosis in MZ patientsin a necropsy study where the presence of PAS-positive material was used as a marker of the Z-gene.Neither immunochemical staining nor determinationof plasma (xi-AT concentrations was performed andtherefore, viewing the occurrence of oxl-AT globulesunrelated to the Z allele in severe disease, one cannotexclude a certain overdiagnosis of the MZ state inthat study. Morin et al.20 have found no increasedfrequency of the MZ phenotype among patients withliver cirrhosis. Blenkinsopp and Haffenden2l find

Table 3 Summary ofcases with immunoperoxidase demonstrable al-AT containing globules in hepatocytes in absence ofthe Zgene

Author (ref) Phenotype PAS-staining Plasma al-A T(g/l) Age (yr) Clinical data

Bradfield et al.116 M 2 * 3 male 79 Acute pancreatitispatient survived 12 days afterbiopsy

Fischer et al.I7t M- 1 8 female - Alcoholic hepatitisno further clinical informationreported

3MS + - no further information reportedKelly et al."8 S - 0 9 male 56 Alcoholic hepatitis

patient survived 5 months afterbiopsy

Present study § M + > 4-0 male 50 Metastatic adenocarcinoma of thepancreas. Patient survived 6 monthsafter biopsy

M 2 * 3 male 67 Severe cardiac decompensation withvenous congestion. Patient still alive

M 2 *7 female 76 Highly active rheumatoid arthritis.Patient survived 7 months afterbiopsy

*Plasma a,-AT determined by immunoprecipitation (reference 2-4 g/l) Pi typing by isoelectric focuLsing.tM-phenotype deduced in absence of family studies, phenotyping done by starch gel and crossed immunoelectrophoresis. Plasma a,-AT by theMancini technique, reference 2 1 g/l.$As for ' with reference for P-a,-AT 1 *8-3 g/l.§Plasma a,-AT determined by electroimmunoassay (reference 0 * 9-1 * 70, average value 1 32 g/l), Pi typing by isoelectric focusing.

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Fig. 1 a1-AT containing globules in hepatocytessurrounding the central vein from the Pi M patient withvenous congestion (Table 3). Immunoperoxidase stain)as described in text x 720.

it probable that Pi MZ is associated with increafibrosis, cirrhosis and hepatocellular carcino(HCC), whereas Kelly et al.22 have found no increin HCC among MZ individuals. In the pres

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prospective series we find a trend towards over-representation of the MZ phenotype among patientswith liver disease but it does not reach statisticalsignificance. It should be stressed that absolutecomparisons are difficult to perform because ofrelatively small sample size and diagnostic hetero-geneity within phenotypic groups. No increase inmean biopsy score for fibrosis or cirrhosis could beseen in patients with phenotype MZ compared toother phenotypes at similar age (Table 2). Of the2 MZ patients having cirrhosis, both had a history ofalcoholism. No MZ patient lacking history ofalcohol overconsumption (3 patients) or cardiacfailure (1 patient) had signs of fibrosis. Interestinglyan increased frequency of CAH was seen in the Pi MSgroup. Fisher et al.17 have reported diagnosis CAHamong 5 of 18 (28%) Pi MS patients with liverdisease. In their study 5/87 (5 - 75 %) of patients withCAH had Pi MS and 18/567 (3- 17%) of all patientswith liver disease had this phenotype. As they haddetermined phenotype only in patients with lowplasma concentrations of cx,-AT, and since axl-ATis often raised in active liver disease,' these figuresmay be an underestimation. Kueppers et al.23 foundthe phenotype MS among 8-5% of patients withchronic active liver disease at the Mayo Cliniccompared to 3 % of healthy blood donors there.

ing These observations together may suggest a predis-position for chronic active liver disease amongpatients with Pi MS. The plasma concentration is

sed very moderately decreased in these individuals and,ma the mechanism behind such an association is un-

ase known.,ent The immunoperoxidase staining as well as PAS-

I #. #...h

Fig. 2 a,-AT globulesin hepatocytes ofa PiMpatient with rheumatoidarthritis (Table 3). al-AT specific immuno-peroxidase staining asdescribed in text x 720.

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staining showed diffuse or granular intracellulara i-AT deposits in a minority of the biopsies studied.This corresponded to slight increases in plasmacx-AT concentrations, total score and inflammatoryactivity seen in biopsies. Our hypothesis has been thatin situations with high plasma ml-AT concentrationsone should expect high intracellular concentrationsreflecting increased synthesis. The regulation ofsynthesis and secretion, however, may well vary indifferent diseased states and the intracellular presenceof precursor proteins, incompletely glycosylatedforms or complexes remains to be studied. Manybiopsies probably contain oci-AT in concentrationsbelow the threshold of sensitivity for the immuno-peroxidase method. In addition, intracellular proteinsmay be extracted or antigenically altered duringtreatment of biopsy sections, possibly giving false-negative immunoperoxidase results.

Biopsies from 102 patients stained positively withthe immunoperoxidase method for intracellularml-AT in leucocytes, Kupffer's cells (30 cases) or

round cells. This may represent degradation productsof complex-bound ocm-AT after phagocytosis butde novo synthesis in these cells, in similarity to thatreported in other cells of histiocytic origin,24 cannotbe excluded.The immunoperoxidase method could correctly

identify intrahepatocellular oci-AT globules in themajority of patients with phenotype MZ and Z, andin that respect was more sensitive than the PAS-staining. Some biopsies from MZ patients had no

visible globules with either method. This is notunexpected due to small sample size. More interest-ing is the occurrence of oci-AT globules in 3 caseswith the normal M phenotype. Plasma proteinanalysis, phenotyping and biopsies were evaluatedindependently in this prospective study of unselectedliver biopsied patients. Thus the chance of findingci-AT globules unrelated to the Z gene can becalculated to be 3/207 or 1 * 4 %.

"Congestive globules"5 are well known and are

usually easily distinguished from "true" oci-ATglobules by localisation and appearance. Our case

with venous congestion (Table 3 and Fig. 1) probablyhad such globules, but they were present periportallyas well as centrilobularly and could not with certaintybe distinguished from al-AT globules. In this case

alone globules stained positively even with antiserafor fibrinogen, IgG, and albumin.

It appears from our cases and those presented inTable 3 that oci-AT globules unrelated to Z geneoccur in elderly or terminal patients with highplasma ocx-AT concentrations reflecting high diseaseactivity. This accumulation may reflect a rate ofsynthesis exceeding the hepatocyte's capacity forglycosylation or other steps in the secretory process.

Carlson, Eriksson, Hdgerstrand

This study was supported by the Elsa and ThorstenSegerfalks Stiftelse.

References

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2 Berg NO, Eriksson S. Liver disease in adults with alphal-antitrypsin deficiency. NEnglJ Med 1972 ;287 :1264-7.

3 Eriksson S, H5gerstrand I. Cirrhosis and malignant hepa-toma in alpha1-antitrypsin deficiency. Acta Med Scand1974;195:451-8.

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Sharp HL. Alpha1-antitrypsin deficiency. Hosp Pract 1971;May:83-96.

6 Jeppsson J-O, Larsson C, Eriksson S. Characterisation ofalpha,-antitrypsin in the inclusion bodies from the liverin alpha1-antitrypsin deficiency. N Engl J Med 1975;293 :576-9.

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8 Lieberman J, Silton RM, Agliozzo CM, McMahon J.Hepatocellular carcinoma and intermediate alphal-antitrypsin deficiency (MZ phenotype). Am J Clin Pathol1975 ;64:304-10.

9Pierce JA, Jeppsson J-O, Laurell C-B. Alpha1-antitrypsinphenotypes determined by isoelectric focusing of thecysteine-antitrypsin mixed disulfide in serum. AnalBiochem 1976;74:227-41.

10 Jensen DM, McFarlane IG, Portmann BS, Eddleston A,Williams R. Detection of antibodies directed against aliver-specific membrane lipoprotein in patients withacute and chronic active hepatitis. N Engl J Med 1978;299:1-7.

11 Taylor CR. Immunoperoxidase techniques; practical andtheoretical aspects. Arch Pathol Lab Med 1978;102:113-21.

12 Ganrot PO. Crossed immunoelectrophoresis. Scand J ClinLab Invest 1972;29, suppl 124:39-41.

13 Kaplow LS. Substitute for benzidine in myeloperoxidasestains. Am J Clin Pathol 1975 ;63 :3.

14 Fagerhol MK. The Pi-system, genetic variants of serumalpha,-antitrypsin. Seriens Haematologica 1968;I, 1:153-61.

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16 Bradfield JWB, Blenkinsopp WK. Alpha,-antitrypsinglobules in the liver and Pi M phenotype. J Clin Pathol1977;30:464-6.

17 Fischer RL, Taylor L, Sherlock S. Alpha,-antitrypsindeficiency in liver disease: the extent of the problem.Gastroenterology 1976;71 :646-51.

18 Kelly JK, Taylor TV, Milford-Ward A. Alpha1-antitryp-sin Pi S phenotype and liver cell inclusion bodies inalcoholic hepatitis. J Clin Pathol 1979 ;32:706-9.

1Eriksson S, Moestrup T, H&gerstrand 1. Liver, lung andmalignant disease in heterozygous (Pi MZ) alpha,-anti-trypsin deficiency. Acta Med Scand 1975;198:243-7.

'I Morin T, Martin JP, Feldman G, et al. Heterozygousalpha,L-antitrypsin deficiency and cirrhosis in adults, afortuitous association. Lancet 1975 ;i :250-2.

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22 Kelly JK, Davies JS, Jones AW. Alpha1-antitrypsin defi-ciency and hepatocellular carcinoma. J Clin Pathol 1979;

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32:373-6.23 Kueppers F, Dickson ER, Summerskill WHJ. Alpha1-

antitrypsin phenotypes in chronic active liver disease andprimary biliary cirrhosis. Mayo Clin Proc 1976;51:286-8.

21 Isaacson P, Jones DB, Judd MA. Alpha,-antitrypsin inhuman macrophages. Lancet 1979 ;ii :964.

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screening for alpha1-antitrypsin globules in an autopsymaterial. Acta Pathol Microbiol Scand [A] 1978;86:325-9.

Requests for reprints to: Dr Joyce Carlson, Departmentof Medicine, University of Lund, Malmo GeneralHospital, Malmo, Sweden.

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Intra- and extracellular'alpha,-antitrypsin in liver with