J. clin. Path. (1962), 15, 291

Needle biopsy of the liver: A reviewSHEILA SHERLOCK

From the Department of Medicine, Royal Free Hospital School of Medicine, London

Specimens of liver for pathological examination maybe obtained during life either by open operation orby one of the various methods of percutaneousneedle biopsy.

Needle biopsy has an interesting history. Firstemployed by Paul Ehrlich in 1883 (quoted byFrerichs in 1884) in a study of the glycogen contentof the diabetic liver, and in 1895 by Lucatello inItaly, it passed into general use in tropical medicinein the diagnosis of hepatic abscesses. In temperateclimates the method did not achieve early popularitybecause of the undoubted risks involved. Just beforeand during the last war, however, there weremany cases of non-fatal hepatitis which stimulatedinvestigators to obtain hepatic material for histo-logical study. Liver biopsy by the needle techniquewas reintroduced among others by Huard, May, andJoyeux (1935) in France, by Baron (1939) in theUnited States, by Iversen and Rbholm (1939) inDenmark, by Axenfeld and Brass-1942)X Gerhnany,and by Dible, McMichael, and Sherlocl\(1943) inBritain. The elaboration of the technique, the'clearerdefinition of indications and contraindications, theintroduction of various safer needles, and especiallythe increase in the number of trained operators, hasnow resulted in needle biopsy being an acceptedprocedure in most large general hospitals. Becausethe method has now become so simple, this does notimply that it is always without risk. Needle biopsyshould always be regarded as potentially fatal. Fivehundred biopsies may be performed without incidentonly for the 501st to be complicated by massiveintraperitoneal haemorrhage demanding immediatetreatment. The patients must therefore be carefullyselected and a real indication for it must be presentbefore the biopsy is performed.

SELECTION AND PREPARATIONOF THE PATIENT

The biopsy should only be performed in hospital andnever undertaken on outpatients.

If the intercostal route is used, the patient must becooperative and able to control his respiration voluntarily.A rent may otherwise be produced in the liver capsule.The method is therefore contraindicated in the confusedor the unconscious.

Special care should be taken before biopsy is under-taken if there is spontaneous bruising or bleeding,especially if the patient is jaundiced. A one-stage pro-thrombin time test should always be performed. If theprothrombin time is prolonged, or even if it is normalbut the patient is jaundiced, vitamin K1 (10 mg. byinjection) should be administered for three days. Iffactor V and VII deficiencies are suspected vitamin KS2(Hoak and Carter, 1961) should be given parenterally.If the level is still increased by more than 2 seconds overthe control and the patient is jaundiced the biopsy shouldnot be performed. Prolongation of the one-stage pro-thrombin time in a patient with a normal serum bilirubinvalue and without a bleeding diathesis is not so serious,and if there are strong indications for the biopsy furthertests of blood coagulation should be performed and ifthese are normal, biopsy can be undertaken. The plateletcount should exceed 100,000.

In every case, the patient's blood group should beknown and 2 pints of compatible blood should be avail-able. Blood transfusion facilities must be adequate in theevent of complicating haemorrhage.A mild sedative, such as 0 2 g. sodium amytal, is given

30 minutes before puncture.If biopsy is required in the presence of ascites, this

should be tapped. If this is not done the surface of theliver will float away from the tip of the biopsy needle anda specimen will not be obtained.

If the liver is small it may not be penetrated by theneedle and distortion may result in puncture of the gallbladder or large blood vessels in the hilum. If there isdoubt concerning the size of the liver, a plain radiographof the abdomen should be a preliminary. If alterationsin the position of the right diaphragm are suspected, achest radiograph should be studied.

POSITION AND LOCAL ANAESTHESIA The patient liessupine with the right side as near the edge of the bed aspossible. A firm pillow may be placed under the left sidein the hollow of the body so that it is slightly tiltedto the right. The right arm is placed behind the head andthe patient looks to the left. After cleansing, the skin isanaesthetized with 2 % procaine solution. A long (8 cm.),fine-bore needle is used to infiltrate the pleura and isthen passed through the diaphragm to anaesthetize theperitoneum and the capsule of the liver. At least 5 ml. oflocal anaesthetic is needed. A preliminary nick is madein the skin with a scapula.

SITE OF PUNCTURE The intercostal technique is on thewhole the more satisfactory for it provides the whole

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transverse depth of the right lobe for the puncture;intra-abdominal viscera are avoided. It does, however,involve puncture of the pleural cavity. The site chosen isthe eighth, ninth, or tenth intercostal space in the mid oranterior axillary line. The place must be the point of maxi-mum dullness to percussion.The subcostal technique is confined to livers enlarged

at least 6 cm. below the right costal margin or in those inpatients with disease of the right chest. It has the advan-tage that a specific lesion on the surface of the liver maybe aimed at and punctured. Otherwise the site chosen isbelow and to the right of the xiphoid process in the mid-clavicular line, the needle being directed to the right andcephalically.

TECHNIQUE OF PUNCTURE

The biopsy may be obtained either by techniques involvingpredominantly aspiration such as the Iversen and Roholm(1939) or Menghini (1958) techniques or by predomin-antly a puncture procedure such as the Vim-Silvermanmethod (Schiff, 1951). All three give good results inexperienced hands. The technique chosen varies with theindividual. The method should be undertaken only bythose well trained in its execution. The novice shouldcertainly practice on the cadaver before attempting it onthe patient and should learn under the supervision of anexpert. Serious complications increase in inexperiencedhands. The aspiration of blood from the liver should notoccasion alarm.

IVERSEN AND ROHOLM METHOD (Sherlock, 1945) Theinstrument used is a handled trocar and cannula modifiedfrom an antrum puncture needle. It is 15 cm. long and1 7 mm. diameter. The needle is passed through the skinbut not through the diaphragm and the patient is theninstructed to 'take a deep breath in, let it out and thenhold your breath'. This displaces the lung upwards andensures apposition of diaphragmatic and costal pleura.The trocar and cannula are now pushed through thediaphragm into the right lobe of the liver. The trocar isnot withdrawn until the instrument is fully a centimetreinside the liver substance. The cylinder of liver tissue isthen punched out by pushing the cannula on a further3 to 5 cm. A 20 ml. syringe is connected to the cannulaand suction is applied and maintained while the cannulais withdrawn. The puncture wound in the skin is sealedwith collodion. The fragment of liver is usually found inthe barrel of the syringe. Occasionally it remains in thecannula.

MENGHINI 'ONE-SECOND' NEEDLE BIOPSY (Menghini, 1958;Norris, Singh, and Montuschi, 1958). The needle used ismade in three diameters, 2, 16, and 1 mm. (Figs. 1 and 2).We use the 1 6 mm. one routinely and reserve the smallestfor poor-risk patients, for instance for those in deepjaundice, where intraperitoneal haemorrhage is possible.The tip of the needle is oblique and slightly convextoward the outside. This results in an excellent cut of thebiopsy specimen without any need to rotate the needle asthe biopsy is performed. The needle also has fitted withinits shaft a blunt nail 3 cm. long, and 0 2 mm. diameter

/

FIG. 1. Lonigitudinal section of the MeAIghiini liver biopsjvneecdle. Note nail in the shaft of the nleedle.Fronti Menghini, 1958.

smaller than the internal diameter of the cannula. Thediameter of the flattened head of the nail is larger thanthat of the cannula and also than that of the internaldiameter of the insertion of the aspirating syringe.This prevents the nail from falling into the cannula andalso from being aspirated into the syringe. This internalblock prevents the biopsy from being fragmented ordistorted by violent aspiration into the syringe andregulates the force of aspiration.The syringe and needle are assembled and 3 ml. of

sterile saline drawn into the syringe. The needle is theninserted through the anaesthetized track down to butnot through the intercostal space and 2 ml. solutioninjected to clear the needle of any skin fragments.Aspiration is now begun and maintained. This is the slowpart of the procedure. With the patient holding hisbreath in expiration, the needle is rapidly introducedperpendicular to the skin into the liver substance andextracted. This is the quick part of the procedure.Rotation is not performed. The tip of the needle is nowplaced under saline in a flat-bottomed glass receptacleand only now is aspiration stopped. It may be necessarygently to inject a little of the remaining saline from thesyringe to free the biopsy.

This method has the advantage that the needle is verybriefly in the liver, only slightly more than the 'onesecond' claimed by the originator; this increases thesafety. The method is also extremely easy to perform.Discomfort to the patient is less than that experiencedwith other techniques. The instrument is simple andtherefore not costly. The main disadvantage is that inthe presence of fibrosis the failure rate is high and thespecimen obtained may be small. Menghini has nowperformed his method on more than 2,000 occasionswithout fatality.

VIM-SILVERMAN METHOD The instrument consists of ashort trocar and cannula which are introduced into theliver. The trocar is removed and the biopsy is punched outby the introduction ofa longer cannula split longitudinally,both halves of which tend to spring apart. The shortcannula is then advanced until the tips of both are level.The apparatus is rotated to break off the end of thebiopsy and withdrawn. Suction is not necessary. Thismethod has a high success rate in the presence of cirrhosis,The sample is large but distortion is often gross.

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FIG. 2. The stages of needle biopsy of the liver using theMenghini technique. (From Menghini, 1958.)1 The biopsy needle with attached syringe is pushed intothe subcutaneous tissue.2 1 ml. of saline is injected to expel any tissue fragmentsfrom the needle.3 While aspiration is maintained and the patient holdshis breath in expiration the needle is quickly insertedthrough the intercostal space and into the liver.4 Aspiration being maintained, the needle is quickly with-drawn.

Other techniques include that evolved by Gillman andGillman (1945) in which the syringe, cannula, and styletare incorporated in one instrument, thus increasing thespeed of manipulation, and the Franseen needle whichresembles the Iversen and Roholm trocar and cannula.

AFTERCARE

After biopsy a small dose of an analgesic, such aspethidine (Demarol), may be necessary to allay discom-fort. A further sedative, such as barbitone soluble 0 75 g.,may be given in the evening. The pulse rate is chartedhourly for the first 24 hours after biopsy, and the physicianshould be called if a rise is observed. Routine visits shouldbe paid four and eight hours after biopsy. A very carefulwatch must be kept on the patient, and if there is any signof haemorrhage the cross-matched blood should beadministered. Absolute rest in bed is essential for 24hours. The patient can then be up and about.During the puncture there may be a complaint of a

drawing feeling across the epigastrium. Afterwards, somepatients have a slight ache in the right side for about24 hours and some complain of pain referred from thediaphragm to the right shoulder. The amount of discom-fort is usually less than that associated with a sternal orlumbar puncture.

DIFFICULTIES

Failures arise in patients with hepatic cirrhosis, especiallywith ascites, for the tough liver is difficult to pierce and afew liver cells may be extracted, leaving the fibrous frame-work behind. Another source of difficulty may be pul-monary emphysema; the liver is then pushed downwardsby the low diaphragm so that the trocar passes above it.

Failure to obtain a sample is often due to the trocarnot being sufficiently sharp to penetrate the capsule. Itshould be sharpened before each biopsy. Alternatively,the trocar may have been withdrawn before the instrumentpierced the liver capsule; the relatively blunt cannula hasgreat difficulty in penetrating even the normal hepaticcapsule.The percentage of successes increases with the diameter

of the needle used but so does the complication rate andone must be weighed against the other. The 1 mm.Menghini needle, for instance, which is extremely safe,often fails to procure adequate hepatic tissue for diagnosis.Thaler (1958) reports too small a specimen for diagnosisin 17% of attempts with the Menghini needle.The question of a second puncture on the same occasion

if the first fails is a difficult one. Provided the patient is notjaundiced, the operator is experienced, and the Menghinitechnique is being used, this is justifiable. Otherwise it isbetter to wait until a subsequent occasion.

LIVER BIOPSY IN PAEDIATRICS

Needle biopsy using the subcostal route has been widelyused with safety in tropical paediatrics to study mal-nutrition (Waterlow, 1948; Meneghello, Espinoza, andCoronel, 1949). It is now being increasingly used ingeneral paediatrics. The Vim-Silverman (Kaye, Koop,

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Wagner, Picou, and Yakovac, 1959) or the Menghini(Hong and Schubert, 1960) techniques may be employed.The Menghini may be the better for it is the quicker. Ininfants local anaesthesia, with 15 to 60 mg. pentobarbital30 minutes before the biopsy, is adequate. The child isrestrained by adhesive strapping across the upper thighsand chest and the subcostal approach used (Kaye et al.,1959). If the liver is small when the intercostal route isemployed the assistant compresses the chest at the end ofexpiration to arrest respiration (Hong and Schubert,1960).

Liver biopsy is particularly useful in investigatingpatients with neonatal jaundice when clinical features areequivocal and biochemistry of little help. An unnecessarylaparotomy, particularly undesirable in a baby withhepatitis, may be prevented. In four patients withneonatal jaundice, a diagnosis was made by this means at3, 6, 8, and 8 weeks of life respectively (Kaye et al., 1959).

In older children general anaesthesia is generallypreferred, depending on the cooperation of the child. Ifsplenic venography is necessary, the two procedures canbe performed under the same anaesthetic (Shaldon andSherlock, 1962).

RISKS AND COMPLICATIONS

In a recent review of 20,016 needle biopsies of theliver the mortality was stated to be 0- 17% (Zamcheckand Sidman, 1953; Zamcheck and Klausenstock,1953) and deaths from haemorrhage were usually inthose patients with a hopeless prognosis.

PLEURISY AND PERIHEPATITiS A brief friction rubcaused by fibrinous perihepatitis or pleurisy maysometimes be heard on the next day. It is of littleconsequence and pain subsides with analgesics. Achest radiograph sometimes shows a small pneumo-thorax.

HAEMORRHAGE Bleeding from the puncture woundusually consists of a thin trickle lasting 10 to 60seconds and the total blood loss is only 5 to 10 ml.Serious haemorrhage is usually intraperitoneal butmay be intrathoracic from an intercostal artery. Inone series significant haemorrhage occurred 16 timesin 7,532 biopsies (0 2%). Laparotomy was requiredin four instances, transfusion alone in three, andexpectant treatment was successful in nine (Terry,1952). The bleeding results from perforation ofdistended portal or hepatic veins or aberrant arteries.In some cases a tear of the liver follows deepbreathing during the intercostal procedure. Severehaemothorax usually responds to blood transfusionand chest aspiration. Haemorrhage is extremely rarein those not jaundiced. It is most common in severehepatocellular disease and where this is suspected,biopsy should be avoided. Patients with obstructivejaundice after vitamin K therapy tolerate liverbiopsy well.

BILIARY PERITON1TIS Using the intercostal approach,the gall bladder should be avoided. There are, how-ever, seven reports of this complication in theliterature, usually in patients with an anatomicalabnormality of the liver (Madden, 1961). Theresultant biliary peritonitis may require surgicaldrainage.A large bile duct in the liver may be punctured in

patients with extrahepatic cholestasis. This has ledto liver biopsy being contraindicated in patientswith obstructive jaundice. In my experience this is avery small risk and the puncture usually seals itselfoff.However, occasionally an infected biliary peri-

tonitis may develop in patients with cholangitis(Terry, 1952). I have never seen this happen althoughoccasionally patients with cholangitis have a rigorand pyrexia after biopsy. Liver material from thesepatients grows coliform bacilli and histology showsan acute cholangitis.

PUNCTURE OF OTHER VISCERA This is very rare. Thecolon may be inadvertently biopsied (Baron, 1939)or the kidney but this is not usually dangerous.

RELIABILITY OF NEEDLE BIOPSIESAS REPRESENTATIVE

It is perhaps surprising that such a small biopsyshould so often be representative of changes in thewhole liver. The lesions of cholestasis, steatosis,virus hepatitis, and the reticuloses are fortunatelydiffuse. This is also true of most cirrhoses, althoughin the coarsely nodular liver of post-necroticcirrhosis it is possible to aspirate a large nodule andfind the architecture normal (Braunstein, 1956). Thefocal granulomatous diseases, such as sarcoidosis,tumour deposits, and abscesses; may be missed butthis is surprisingly infrequent. In a recent comparisonof the results of needle biopsy compared withoperative biopsy there was complete agreement in76-9 %, minor differences in 16 1 %, and in only 7%did needle biopsy completely miss the lesion(Federlin and Sandritter, 1961). Liechty (1958), onthe other hand, reports 40% positive diagnoses withthe needle method compared with 98-3 % using openbiopsy. Most authors, however, comment on theincrease in fibrous tissue under the capsule inoperative biopsies which gives a false impression ofthe liver as a whole (Popper and Schaffner, 1957;Sherlock, 1958). Also operative biopsies may showartefactual changes such as patchy loss of glycogen,haemorrhages, and polymorph infiltration, all due tothe effects of the operation and of the anaesthetic.The combination of peritoneoscopy with needle

liver biopsy enables the surface of the liver to be

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visualized and the biopsy taken directly from anyfocal abnormality (Becker, 1961; Samuelsson andSjostedt, 1961). More than one biopsy may be taken.The specimen is subcapsular and may therefore befibrotic and again not representative of the interiorof the liver. In experienced hands such as those ofCaroli, Mainguet, and Ricordeau (1961) in France,or Kalk and Wildhirt (1962) in Germany, peritoneo-scopy adds little to the time taken or to the discom-fort involved. With less practised operators, however,the increase in extent of the procedure does notcompensate for the information obtained.

NAKED-EYE APPEARANCES

A satisfactory biopsy is 1 to 4 cm. long and weighs50 to 200 mg.The cirrhotic liver tends to crumble into fragments

of irregular contour. The fatty liver has a pale,greasy look and floats in the formol saline fixative.The liver containing malignant deposits is a dullwhite. The liver from a patient with Dubin-Johnsonhyperbilirubinaemia is diffusely chocolate coloured(Fig. 3).

Terry (1954) emphasized the value of detailedexamination of the biopsy. In cholestatic jaundice,the greenish central areas contrast with the lessgreen periphery of the lobule. Transilluminationmay show up granulomata, for instance, in sarcoi-dosis or tuberculosis as translucent areas. Thevascular centres of lobules in hepatic congestion maybe obvious.

FIXATION AND PREPARATION OF SPECIMEN

The specimen is less likely to fragment, and easier to cutin one plane, if it is laid on a strip of blotting paper beforeplacing in 10% formol saline. The receptacle used shouldbe a wide-mouthed one so that it is easily introduced andremoved. The biopsy is processed in the usual way on aHistokine automatic preparative machine using a 21-hourcycle and including formol mercury as a post-fixative.After embedding in paraffin, sections are cut of 5,uthickness.

FIG. 3. Paraffin blocks of needle liver biopsies from anormalpatient and (below) the chocolate coloured biopsy ofa patient with Dubin-Johnson hyperbilirubinaemia.

Haematoxylin and eosin and reticulin by a silvermethod are routine stains, and to most sections Perlsstain for iron and periodic-acid-Schiff for glycogen arealso applied.

Serial sections are important for the diagnosis of lesionswhich may be scattered through the liver.

INDICATIONS FOR NEEDLE BIOPSY ANDHISTOLOGICAL APPEARANCES

NORMAL APPEARANCES The portal zones bear aregular relation to the central areas. This may bedifficult to establish in small biopsies, especially ifno portal zones have been obtained, but thisorientation is an essential preliminary before report-ing the biopsy. Each portal zone consists of one ortwo bile ductiles, a branch of the hepatic artery andone of the portal vein, a few mononuclears, and anoccasional fibroblast. The liver cell plates are onecell thick and contain abundant glycogen. Mitosesare not seen in the liver cells, which are usuallymononucleate and of regular size. The sinusoids arelined by Kupffer cells and can be seen convergingupon the central vein.

JAUNDICE Acute jaundice rarely merits liver biopsyfor clinical observation and biochemical methodswill usually make the diagnosis certain. The methodcarries extra risk in this group.

Acute virus hepatitis In acute virus hepatitis(Fig. 4) the lobular pattern is usually maintainedintact, although if necrosis is massive the reticulincollapses (Dible et al., 1943). The portal zones showa widening composed mainly of mononuclears,polymorphs, and eosinophils, fibroblasts, and proli-ferating bile ducts having a slit-like lumen. Thisportal lesion is very prominent and some cellularinfiltration and scarring may be observed for monthsafter apparent clinical recovery. The sinusoids areinfiltrated with mononuclears. Hepatocellularnecrosis is maximal in the centrizonal areas. Focaleosinophilic necrosis with acidophilic bodies in theliver cells may also be seen. Bile thrombi, maximallycentrizonally, are noted, especially in the Kupffercells and liver cells.

Cholestatic jaundice In cholestatic jaundice theconstant characteristic feature is a normal lobulararchitecture with bile retention, maximal centri-zonally in bile canaliculi, liver cells, and in Kupffercells. The extent of the bile retention depends on thedepth of jaundice and the duration, and bile plugspersist for some time after the jaundice fades. Mildpatchy necrosis and nuclear changes may be seen inthe centrizonal liver cells.Imposed on these background features are certain

changes, mainly in the portal zones, which depend

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Jaundice

FIG. 4. Acute virus hepatitis. The centrizonal area shows loss ofliver cells. Theportalzone is heavily infiltrated with mononuclears and this cellular infiltration extendsthrough the sinusoids towards the central vein. The general architecture of the liveris maintained intact. Stained haematoxylin and eosin x 75.

FIG. 6

FIG. 5. Extrahepatic biliary obstruction due to gall stones. The zonal architecture of the liver is maintained intact. Theportal zones show expansion and contain proliferating bile ducts, fibrosis, and cells. The centrizonal area shows heavy bileaccumulations in liver cells and bile canaliculi. Surrounding liver cells show variation in size and in nuclei. Stainedhaemato-xylin and eosin x 72.FIG. 6. Same case as Fig. 5. The expanded irregular portal zone shows numerous proliferated bile ducts some of themhaving a wide lumen. The cellular infiltrate is mononuclear with many polymorphs, especially around the dilated bile ducts.Stained haematoxylin and eosin x 275.

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FIG. 7 FIG. 8FIG. 7. Cholestatic jaundice following chiorpromazine. The zonal architecture is maintained intact. The centrizonal areashows heavy bile stasis. Liver cells, especially centrizonally, show patchy, often feathery, necrosis with evidence ofregenera-tion shown by variation in cell and nuclear size. The portal zones are virtually normal. Stained haematoxylin and eosin x 72.FIG. 8. Same case as Fig. 7. Portal zone shows a slight increase in cellularity, mainly mononuclear. A few eosinophilscould also be demonstrated. Stained haematoxylin and eosin x 275.

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on whether the jaundice is of intrahepatic or extra-hepatic origin. The essential difference in the liverbetween extra- and intrahepatic biliary obstructionis the dilatation of bile ducts in the one and not inthe other. This may proceed in extrahepatic chole-stasis to rupture of small ducts within the liver andthe production of so-called 'bile lakes' or 'bilenecroses' which are diagnostic of a block in largebile ducts. Unfortunately, these are not constantlyseen. Reduplication of bile ducts having a widelumen and sometimes containing bile is a feature ofextrahepatic cholestasis (Fig. 5 and 6). Invasivefibrosis in the portal zones with predominantpolymorph infiltration also suggests extrahepaticcholestasis.

In intrahepatic cholestasis due to virus hepatitis,necrotic changes in the liver cells are prominent andthe histological picture is usually diagnostic (Shaldonand Sherlock, 1957).

In intrahepatic cholestasis due to drugs of thechlorpromazine type (Fig. 7 and 8) the portal zonesshow mild cellular infiltration, mainly histiocytic,but in the early stages eosinophils may be prominent.Liver cell damage tends to be more prominent, withfeathery degeneration and ballooning of cells,peripheral vacuolation, and hyaline deposits. Focalnecrosis of liver cells may be seen. Anisocytosis ofliver cells and mitoses are frequent (Read, Harrison,and Sherlock, 1961).

In intrahepatic cholestasis due to drugs of themethyl testosterone or norethandrolone type theportal zones are normal (Schaffner, Popper, andChesrow, 1959).

Hepatic histology cannot be relied upon todifferentiate with certainty cholestasis of intra-hepatic from that of extrahepatic origin (Popper andSchaffner, 1959).

Haemolytic jaundice In haemolytic jaundice,hepatic histology is normal apart from increased irondeposits in Kupffer and liver cells.

Congenital hyperbilirubinaemias In the usualGilbert variety hepatic histology is normal (Krarupand Roholm, 1941). In the Dubin-Johnson type(Dubin, 1958) the liver cells are filled with a brownpigment believed to be a lipofuscin, 'black liverjaundice' (Fig. 9).

THE CIRRHOSES Needle biopsy is not necessary inthe patient with advanced cirrhosis in whom diag-nosis is quite obvious clinically. It is useful in recog-nizing well-compensated cirrhosis as a cause ofhepatomegaly or splenomeagaly or to obtain abaseline before some treatment, such as withcorticosteroids, is begun.

Cirrhosis is an anatomical diagnosis which shouldbe confined to lesions showing the following

characteristics (Report on the classification andnomenclature of cirrhosis of the liver, 1956).

1 All parts of the liver must be involved, althougnnot necessarily each lobule; this allows the inclusionof the post-necrotic type in which large nodules oftencontain one or more intact lobules.

2 Hepatocellular necrosis must be present atsome stage; this allows the inclusion of cirrhosiswhich, at the time of histological examination,does not show damage to hepatic cells.

3 Nodular regeneration must be present. Fibroustissue bands join central veins with portal tracts andso disorganize the normal hepatic architecture.The biopsy from the cirrhotic liver is liable to be

small and fragmented and it may be very difficult,not only to be certain that the normal architectureis lost, but also to differentiate between a fine'portal' cirrhosis and a coarse 'post-necrotic' one.The term cirrhosis should never be used synony-mously with fibrosis.

Cirrhosis of the alcoholic is invariably fatty and inthe acute stage focal necrosis of liver cells withsurrounding polymorph reaction may be seen(Fig. 13). The hyaline deposits in liver cells describedby Mallory are best demonstrated by the phos-photungstic-acid-haematoxylin method or by luxolfast blue when the hyaline stains dark purplish blue(Becker and Treurnich, 1959). They are not specificfor alcoholism (Popper, Rubin, Krus, and Schaffner,1960). In the alcoholic, various stages short ofcirrhosis, usually stellate fibrosis may be noted andthe cellular reaction may also be extremely acute(Beckett, Livingstone, and Hill, 1961).An increasing number of patients, usually but not

necessarily young, and women rather than men,with an acute 'juvenile' cirrhosis are being seen. Thepatients are usually jaundiced and show high serumgamma globulin values. An autoimmune process inthe liver has been postulated and L.E. cells maysometimes be found in the blood (Mackay, 1961).The condition may in some instances be initiated byacute virus hepatitis. Liver biopsy shows a veryactive cirrhosis of post-necrotic type with isolationof groups of liver cells by young fibrous tissue(Fig. 10). The liver cells may be giant, multinucleate,or form rosettes. The biopsy is heavily infiltratedwith lymphocytes and plasma cells (Fig. 11).Haemochromatosis may be suspected by the brown

pigment seen in the liver in haematoxylin and eosinstained sections. The presence of excess iron shouldalways be confirmed by Perls' reaction. Siblings ofpatients with haemochromatosis may show in-creased amounts of iron in the liver (Williams andScheuer, 1962) and this should always be consideredin a patient who shows unsuspected iron deposition.Study of 558 consecutive liver biopsies showed

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FIG. 10 FIG. 1I1FIG. 10. Acute juvenile'cirrhosis. The lobular architectutre FIG. 11. Same case as Fig. 10. Reticulin stains confirmis completely disturbed. Isolated groups of liver cells, which the isolation of liver cells by bands offibrous tissue. x 120.often assume a rosette-like appearance, are separated bythe septa of connective tissue. Remaining cells are large A-'with clear cytoplasm. Lymphocytic and plasma cell > N N t

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'9,. lSLAflt-i.JFIG. 13. Cirrhosis of the alcoholic. The picture is of a fineportal type cirrhosis. Liver cells show gross fatty changeand fatty cysts are sometimes seen. Foci of inflammatorycells are scattered through the parenchyma. Stainedhaematoxylin and eosin x 120.

FIG. 12. Hepatolenticular degeneration (Wilson's disease). FIG. 14. Primary biliary cirrhosis. The lobular pattern isLiver cells adjoining a fibrous tissue band show gross grossly disturbed by fibrous bands which extended fromvacuolation of their nuclei (glycogenic degeneration) and the portal zones. The fibrous septa contain aggregations ofoccasionally fatty change. Haematoxylin and eosin x 165. lymphocytes. Stained haematoxylin and eosin x 40.

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significantly a higher proportion of iron in thosewith hepatic disease (44%) than in those withoutsignificant disease (Zimmerman, Chomet, Kulesh,and McWhorter, 1961).

Wilson's disease Hepatolenticular degeneration(Wilson's disease) should be considered in anypatient with cirrhosis presenting under 30 years oldeven if there are no abnormal neurological changes.Fatty change and peripheral nuclear vaculation ofthe liver cell (Fig. 12) in a young patient with post-necrotic cirrhosis is very suggestive (Anderson andPopper, 1960). If this diagnosis is being considereda small portion of the liver biopsy should be fixedimmediately in a freshly prepared solution ofrubeanic acid in 70% alcohol when the copper is seenin sections as brownish-black granules in the livercells (Uzman, 1957).

Biliary cirrhosis Patients with prolonged chole-stasis, whether intrahepatic or extrahepatic, ulti-mately develop biliary cirrhosis. There is character-istically a widening of the portal areas by fibroustissue which encroaches on the lobules and sendssepta into the parenchyma. Nodular regeneration isnever so prominent as in other forms of cirrhosis.In the later stages, difficulties may arise in differ-entiating this from other types of cirrhosis.

Secondary biliary cirrhosis follows prolongedextrahepatic biliary obstruction often due totraumatic stricture. If the biopsy is taken during anepisode of cholangitis Gram-negative organismsmay be cultured from it and this should always beattempted in such patients. Polymorphs are con-spicuous in the portal zones where bile ducts areproliferating widely.

In primary biliary cirrhosis the aggressive in-filtrating fibrosis in the portal zones is particularlymarked and bile ductules are inconspicuous (Fig. 14).Lymphocytes may form foci and plasma cells maybe obvious. However, the picture of primary biliarycirrhosis may be very difficult to distinguish fromsecondary biliary cirrhosis in the individual case(Sherlock, 1958).

HEPATIC TUMOURS Although Fisher and Faloon(1958) believed that needle biopsy was particularlyhazardous in the presence of metastatic malignancy,the mortality of their series being 12%, others statethat the risk is no greater than in other patients(Parets, 1959; Fenster and Klatskin, 1961), and thishas been my own experience. The chance of obtain-ing a positive biopsy increases the greater the sizeof the liver. The percentage of positive biopsies inrecent large series has been 64 to 78 (Ward, Schiff,Young, and Gall, 1954; Parets, 1959; Nelson andSalvador, 1960; Fenster and Klatskin, 1961). If acylinder of tissue is not obtained, it is worth centri-

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FIG. 15. Hepatic metastasisfrom primary in bronchus. Atthe top of the section relatively normal liver tissue canbe seen infiltrated by mononuclear cells. The metastasis isseparated by a zone offibrous tissue. The malignant cellsare undifferentiated. Stained haematoxylin and eosin x120.

fuging any debris or blood clot and examining it formalignant cells. Study of sections will not alwaysenable the site of the primary to be detected,especially if the tumour is undifferentiated (Fig. 15).

THE RETICULOSES Care must be taken not to attemptto biopsy if there is any bleeding tendency.

In Gaucher's disease the typical Gaucher cell maybe readily shown in the liver.

It is rare for liver biopsy to help in the diagnosisof multiple myeloma.The liver is involved in about 70% of patients

with Hodgkin's disease (Levitan, Diamond, andCraver, 1961) and it is therefore worthwhile per-forming liver biopsy where histological proof cannototherwise be obtained. A positive result is, however,not constant and in one series none of 10 patientswith Hodgkin's disease gave a positive liver biopsy,although in all the liver was at necropsy shown to beinvolved (Amos and Goodbody, 1959). In anotherseries, eight of 12 patients with leukaemia and five

Sheila Sherlock300

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Needle biopsy of the liver: A review 0

of 30 with other lymphomas gave positive results(Nelson and Salvador, 1960). In many instances,the picture is a non-specific one of mononuclearinfiltration in the portal zones. When the result ispositive, the Hodgkin's or leukaemic tissue ismaximal in the portal zones, and in myeloid leuk-aemia immature cells of the white cell series can beseen crowding the sinusoids.

In the myeloproliferative disorders, liver biopsymay show sinusoidal infiltration by multinucleatedcells resembling megakaryocytes, discrete sinusoidalfoci of haemopoietic tissue, or diffuse infiltration byprimitive myeloblast-like cells (Amos and Goodbody,1959).

GRANULOMAS OF THE LIVER The granuloma is amiliary lesion comprising central amorphous debrissurrounded by a zone of epithelioid cells with orwithout giant cells, lymphocytes, and fibrous tissue(Fig. 17). It is non-specific and the observation ofsuch a lesion in a liver biopsy section is diagnosticof a granulomatous disease but not of any specificone.

If granulomatous disease is suspected, and thefirst sections give negative results, serial sectionsmust be examined from the entire biopsy.A granulomatous lesion will be found in the liver

in 60% of patients with sarcoidosis but there mustbe a compatible clinical picture and the Kveim testshould also be performed (James, 1961).

If miliary tuberculosis is suspected, the biopsyshould also be cultured (Healey, Leff, and Rosenak,1959) and Ziehl-Neelsen stains should be applied.The centre of the granuloma may show caseation. Ina series of 64 biopsies on patients with tuberculosis,II % showed miliary granulomata (Singh, Jolly, and

4~~~~~~~~~~~~~~~4

K '*!A5 A-v ;

!f 5~~~~~~~~~~~~~~~~~~~~1FIG. 16. Sarcoidosis. A well-demarcated granuloma isseen with surrounding lymphocytes and central, pale-staining epithelioid cells. Stained haematoxylin and eosinx 84.

Singh, 1960) and in another 80% of 30 patients withextrapulmonary tuberculosis, gave positive liverbiopsies (Korn, Kellow, Heller. Chomet, andZimmerman, 1959). Miliary granulomas may befound in the liver after B.C.G. vaccination (Camain,Senecal, and Couturier, 1956).

In brucellosis, granulomata may be scatteredthroughout the liver. The lesions tend to be smallerthan in sarcoidosis and less clearly demarcated.Both Br. abortis (Cohen, Robins, and Lipstein, 1957)and Br. melitensis (Fogel and Lewis, 1960) have beencultured from liver biopsy specimens.

Hepatic granulomata may also be seen in lepro-matous leprosy and acquired secondary syphilis.Similar lesions are seen in histoplasmosis (Parsonsand Zarafonetis, 1945) and in coccidioidomycosis(Ward and Hunter, 1958), and in both conditionsappropriate stains for the organism should beapplied and the biopsy should be cultured.

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FIG. 17. Pyogenic liver abscess secondary to suppurativeappendicitis. The portal zones show a dense infiltrationwit/i cells, mainly polymorphionuclear. Stained haema-toxylin and eosin x 75.

INFECTIONS Accumulations of polymorphs in theportal zone may be found near a pyogenic liverabscess (Fig. 17). If this diagnosis is suspected, the

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biopsy should be stained by Gram's method and asmall portion cultured.

In leptospira icterohaemorrhagica infection, theliver biopsy shows focal necrosis and periportalinfiltration with histiocytes and polymorphs. Theleptospira can sometimes be demonstrated andcultures may reveal the organism (Ostertag, 1950).

In hepatic amoebiasis, the changes are usuallynon-specific with polymorph and monocytic in-filtration of the portal zones. Small abscess cavitiesmay be punctured.

Suspected hydatid disease is a contraindication toneedle biopsy as this may lead to fatal anaphylaxisor peritoneal dissemination.

METABOLIC DISORDERS A death has been reportedfollowing liver biopsy in a patient with advancedamyloidosis (Volwiler and Jones, 1947) and it isbelieved that the liver may be easily split by thebiopsy needle in this condition. In another series,intraperitoneal haemorrhage was found in one of18 patients with amyloidosis after liver biopsy(Stauffer, Gross, Foulk, and Dahlin, 1961). In myexperience, the risk of haemorrhage is no greater

than in other conditions and this is justified by theadvantage of positive diagnosis.

Secondary amyloidosis is shown by the depositionof amorphous pink-staining material between thesinusoids and the liver cell columns, which arenarrowed (Fig. 18). The nature of the material maybe confirmed by methyl violet or Congo red stains.In the primary form the amyloid material is depositedin the walls of hepatic arterioles.The liver from a patient with glycogen disease is

packed with glycogen but no more so than in manynormal subjects. Increase in the stability of theglycogen must also be shown. If possible, a portionof the biopsy, deep-frozen, should be sent to a centrecapable of performing enzyme studies on it.

CONGENITAL HEPATIC FIBROSIS This condition isusually diagnosed in childhood and must be carefullydistinguished fromjuvenile cirrhosis (Kerr, Harrison,Sherlock and Walker, 1961). The lobular structure isintact but each lobule is surrounded by dense mature,

FIG. 19. Congenital hepatic fibrosis. The liver lobule issurrounded by a dense band of mature, avascular fibroustissue. The connective tissue band contains numerous bileducis, some of which contain bile. Portal vein radicles areinconspicuous. Stained Best's carmine x 160.

FIG. 18. Secondary amyloidosis. The liver cell columnsare compressed by pale-staining amorphous tissue. Stainedhaematoxylin and eosin x 275.

fibrous tissue in which bizarre-looking bile ducts areprominent (Fig. 19). Portal vein radicules aredifficult to find in the fibrous tissue bands and thishypoplasia may account for the portal hypertensionwhich can be a presenting feature. The condition insome instances seems to be a variant of polycysticliver and the kidneys may occasionally also beinvolved.The liver is so tough that needle biopsy may fail

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Needle biopsy of the liver: Areview3

FIG. 20. Accidental carbon tetrachloride poisoning. To theright ofthe section liver cells are necrotic andshow hydropicdegeneration and fatty change. Surviving liver cells to theleft of the section show occasionalfatty change. The portalzones are unaffected.

or the sample may be so small that accurate diagnosisis impossible. In these circumstances open hepaticbiopsy may be necessary.

CHEMICAL LIVER INJURY

The best example of chemical injury to the liver ispoisoning by carbon tetrachloride which may besuicidal or accidental. In general, in all chemicalpoisonings the kidney suffers more than the liverand anuria is a greater risk than hepatic failure.

Liver biopsy in this group shows a centrizonalnecrosis with hydropic change in the liver cellswhich show clear cytoplasm and pyknotic nuclei.Swelling of surrounding liver cells and fatty changeis conspicuous. The portal zones are unaffected(Fig. 20). Cellular reaction is minimal in the earlystages but later polymorphs may be found inrelation to the centrizonal necrotic cells. The pictureis quite different from infective hepatitis where theportal cellularity is conspicuous and fatty change isabsent.

NEEDLE LIVER BIOPSY IN CLINICAL RESEARCH

Histochemical tenchiques have been particularlyapplied to various enzymes. For instance, non-

specific alkaline and acid phosphatase can bereadily demonstrated in liver biopsy sections (Sher-lock and Walshe, 1947; Barka, Schaffner, andPopper, 1961). Bile canaliculi may be beautifullyshown by staining for adenosine triphosphataseand staining for glucose 6-phosphatase may be usedin suspected glycogen disease (Wachstein, 1959).Electron microscopy is being increasingly used to

show structural changes in liver biopsy materialwhich are not obvious by light microscopy (Novikoffand Essner, 1960; Popper and Schaffner, 1961).Intracytoplasmic particles, perhaps the actual virus,have been shown in liver cells in infective hepatitis(Braunsteiner, Fellinger, Pakesch, Beyreder, Grab-ner, and Neumayr, 1957) and sinusoidal abnormal-ities also noted in the early stages of this condition(Cossel, 1959). Alterations in the microvilli lining thebile canaliculi have been seen in various forms ofcholestatic jaundice including that produced bynorethandrolone (Schaffner and Popper, 1959;Schaffner, Popper, and Perez, 1960). Electron micro-scopy may be useful in detecting abnormalities inpigment deposition within the liver in the siblingsof patients with haemochromatosis (Scheuer andWilliams, 1962).

Electron microscopy may be combined withhistochemistry, the 'macromolecular cell pathology'of Novikoff and Essner (1960). Adenosine triphos-phatase is localized to the microvilli of the canaliculiand 5-nucleotidase to the microvilli of the sinusoidalborder (Popper and Schaffner, 1961). Acid phos-phatase is found in Kupffer cells, degenerating foci,and regenerating nodules; alkaline phosphatasedefines cholangioles (Novikoff and Essner, 1960).This so-called macromolecular method offers excitingpossibilities for future research.

Quantitative analysis of liver biopsy specimens ismade inaccurate by sampling difficulties and byfailure to find a suitable standard of reference. Inthe liver with normal structure, results are reason-ably reliable. For instance, the glycogen content ofthe diabetic liver has been shown to be normal(Hildes, Sherlock, and Walshe, 1949) and analysisof needle biopsy material gives a fair indication ofthe fat content of the diffusely fatty liver (Billing,Conlon, Hein, and Schiff, 1953). Difficulties arise,particularly in biopsies from cirrhotic livers wherethe proportion of fibrous tissue is uncertain.Chemical analysis for D.N.A., which is confined tothe nucleus, probably represents the best referencebase (Kosterlitz, 1958), although this may be value-less where the proportion of cells of different typesis variable. Alternatively the substance being investi-gated may be referred to dry weight or to totalnitrogen content of the biopsy.Many quantitative studies of hepatic enzymes have

been made on needle biopsy material. Waterlow(1958) has shown that in malnutrition cholinesterasewas reduced in activity but other enzymes werewell preserved. Nikkila and Pitkanen (1959) havestudied 14 enzymes in samples removed by needlebiopsy from patients with thyrotoxicosis. Schmidt,Schmidt, and Wildhirt (1959) in Germany andRyser, Frei, and Vannotti (1958) in Switzerland

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have also made extensive quantitative studies on

hepatic enzymes in many disease states. Recently,Pitney and Onesti (1961) have described a m2thodfor the microbiological assay of vitamin B12 andfolic acid concentrations in needle biopsy specimens.

Although these results of microchemical analysisare of considerable interest, the problem of thesampling error has not been solved and in patholo-gical liver biopsies this has, to date, afforded an

insuperable obstacle to scientific progress.

I am greatly indebted to Professor K. R. Hill, to Dr. P.J. Scheuer, and to thie technical staff of the PathologyDepartment of the Royal Free Hospital for much helpfuladvice. 1 wish also to thank Mr. R. R. Phillips and thestaff of the Photographic Department for help withthe illustrations.

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