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FINE-NEEDLE ASPIRATION BIOPSY OF RAT LIVER: TECHNIQUES AND APPLICATIONS
CHAPTER 4
FINE-NEEDLE ASPIRATION BIOPSY OF RAT LIVER: TECHNIQUES AND APPLICATIONS U. BOELSTERLI AND G. ZBINDEN
1 Introduction It is now generally accepted that the fine-needle aspiration biopsy (FNAB) method which is widely used in clinical medicine yields reliable and reproducible results in the cytodiagnosis of human tumors (chapter 2 in this book). The complication rate of the procedure is low. Thus, there are very few contraindications. Furthermore, the biopsy technique is simple and rapid, and can be performed repeatedly.*®Considering these favorable cUnical experiences, it was logical to try to adapt this method for cytological investigations in small laboratory animals.^® In experimental toxicology, the objectives of the studies differ to some extent from those in clinical pathology. The clinician is primarily interested in the diagnosis of tumors and in assessing their dignity. In experimental studies the aim of the method is to recognize in the living animal a variety of pathological alterations which can be caused by chenucal substances. Although FNAB sampling can not always replace conventional histology, the advantages of the method are obvious: first, it is not necessary to sacrifice an animal just to get a small piece of tissue. An even more important advantage is the possibility of monitoring individual animals by serial punctures over a long period of time. In this way, individual biological variations can largely be eliminated. A biopsy is, of course, not always representative of the whole organ. A certain sampling error must therefore be taken into consideration.*^ The importance of this error varies according to the nature of the lesion: a diffuse lesion is much more likely to be identified than one which is focal. In order to increase sampling accuracy, it is recommended to take biopsies from several sites of the organ and to perform two FNAB at the same time.* In small laboratory animals, an organ which is to be punctured successfully has to meet the following requirements: it must be within easy reach for blind puncture or should be accessible to palpation. Furthermore, it should represent a relevant target for toxic effects. From this it is clear that the Hver was the first organ for which the FNAB technique was developed. 2 Uver FNAB in the Rat The rat liver is composed of five lobes: the right medial ( = right accessory) lobe, the left medial ( = left accessory) lobe, the right lateral lobe, the left lateral lobe, and the caudate lobe ( = papillary process of the right lateral lobe).*^ In order to avoid aspiration of gastric content, biopsies are taken from the right side of the liver. Care must be taken to puncture always the same lobes, i.e. the right medial and right lateral 17
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υ . BOELSTERLI AND G. ZBINDEN
liver lobes (Fig. 1). Since the rat lacks a gall bladder the risk of bile peritonitis is minimal. Routinely two punctures should be performed at the same time. Long-term experiments showed that even repeated liver FNAB did not affect hepatic structure and function." No deaths due to the punctures have, so far, been recorded. The slight bleeding into the peritoneal cavity that occurs after a puncture is of no consequence. The biopsy channel in the liver is replaced by fibrotic tissue containing a few macro phages with hemosiderin inclusions.
FIG. 1. Site of puncture in the rat liver. The xiphoid process (px) of the sternum is palpated, and the needle is inserted into the right medial liver lobe (rm) and the right lateral Uver lobe (rl). The needle should be in an angle of approx. 45** to the body surface, and attention should be paid to not penetrating too deeply with the needle nor to pierce the diaphragm (d). For details see text (p. 19).
2.1 ANESTHESIA AND PRELIMINARY TREATMENT
Anesthesia. The rat is given a slight ether narcosis and is placed with the dorsal side on an operating table where its limbs are slightly stretched and fixed (Fig. 2). During further manipulations, anesthesia is maintained by supplementary ether inhalation. So far no change in hepatocyte morphology or changes in the enzyme activity pattern have been recorded due to the very short and slight ether narcosis (c. 30 sec). Preliminary treatment is not necessary. There is no need to shave the ventral side of the rat. Since further processing of the aspirated material must be done very quickly, it is important that all necessary equipment (glass slides, fixatives) are in easy reach. 2.2 TECHNIQUE OF LIVER FNAB
Equipment. A handle for disposable syringes (Cameco, M. Bieri, CH-3097 LiebefeldBern, Switzerland), 20-ml plastic syringes (Luer type) and 22-gauge needles (0.7 mm in diameter) are necessary. The syringes are not filled with buffer solution and should be absolutely dry.
Fine-needle Aspiration Biopsy of Rat Liver 19 Technique (Figs. 1 and 2). The xiphoid process of the sternum is palpated. The needle is inserted at an angle of approx. 45° at the caudal right side of the xiphoid process. Deep penetration must be avoided. When the needle has entered the right medial and lateral liver lobes, the piston of the syringe is retracted and, while maintaining negative pressure, the needle is moved back and forth 2-3 times by about 1 cm. In this phase the needle is directed into different areas of the lobes. The piston is then released and the aspiration is interrupted. The needle is quickly retracted. It is disconnected from the syringe. The syringe is filled with some air and reconnected to the needle. The aspirated material is expelled immediately either on a glass sUde to prepare a smear or into a buffer or fixative solution (p. 20).
FIG. 2. Fine-needle aspiration biopsy of rat liver. The animal is in slight ether narcosis and fixed on an operating table. The biopsy is performed percutaneously with a fme needle (0.7 mm in diameter), attached to a 20-ml syringe. Puncture and aspiration is facilitated by using a special handle. The other hand thereby is free for palpating.
2.3 PROCESSING OF THE BIOPSY SPECIMEN
In contrast to conventional liver biopsy with the needle described by Menghini^' where a cylinder of hver tissue is obtained which can be fixed and further processed for histological studies, the FNAB technique yields a number of large and small tissue fragments. By one puncture one usually obtains 5-10 mg liver tissue which is sufficient for many quantitative biochemical assays. During aspiration, a varying amount of blood is admixed. The biopsy specimens can be treated by two different ways: (1) they are smeared on glass sUdes, fixed and stained with routine cytological methods or undergo cytochemical staining reactions for microscopic evaluation; or (2) they are washed free from blood in buffer solutions and collected either for homogenization for quantitative biochemical assay or undergo special treatment for fixation or enzymatic dissociation (p. 27).
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υ . BOELSTERLI AND G. ZBINDEN
2.3.1 Preparation of Smears For these preparations care should be taken not to aspirate too much blood. The material in the needle is expelled onto one or several glass sUdes by depositing a single drop. Usually the tip of the syringe contains valuable material which can also be blown out onto further sudes. A smear is prepared immediately, using a second glass slide (Fig. 3). It is essential for good staining quality that the preparation of the smear and fixation is performed before the blood has coagulated.
FIG. 3. Technique of smear preparation: (a) a single drop of the content in the needle or in the tip of the syringe is deposited on a clean glass slide; (b) it is immediately covered with a second glass slide, and with a slight press a smear is quickly prepared; (c) clusters of hepatic tissue are spread over the slide, intermingled with blood. Monolayers of hepatocytes should be preferred for microscopical examination.
2.3.2 Washing of the Samples In order to wash the biopsy samples free from blood (which is usually admixed during aspiration), the content of the needle and tip of the syringe is expelled into icecold phosphate-buffered saline (PBS)^^(I). Specimens are collected by means of a small syringe and subsequently washed by repeated transfers into fresh PBS. Other methods^" are also described to concentrate and process aspirated cells from tumors (FicoU gradient and cytocentrifuge). (7) PBS: 8.00 g NaCl, 0.20 g KCl, 1.15 g Na^PO^ 11 water (pH 7.4).
0.20 g KH^O^
dissolved in
2.3.3 Homogenization The biopsy samples are transferred into a microhomogenizer glass tube (Duall, 0.3-1.0 ml, Kontex, Vineland, NJ, USA) and homogenized in 0.3-0.6 ml PBS (ice-cold) with a teflon pestle either by 10 strokes (1500 rpm) or, even better, by manual rotating up-and-down movements (on ice). Enzyme assays are usually performed on the crude homogenate, since differential centrifugation of the homogenate would be very dif ficult. The homogenate is stirred each time before aliquots are taken. However, crude liver homogenates obtained by FNAB in mice were compared with freeze-dried samples which were shown to yield higher activities for some enzymes. ^ Moreover, a method for subcellular fractionation of biopsy specimens by density gradients was recently describ ed.^ 3 Cytology For cytological studies of FNAB smears, two staining techniques are routinely used: (1) Papanicolaou staining, and (2) May-Grünwald-Gíemsa staining." The two methods are compared in Table 1. In our laboratory, we almost exclusively use the Papanicolaou (PAP) staining for liver cytology which gives more representative and reliable results.
Fine-needle Aspiration Biopsy of Rat Liver
21
TABLE L Comparison of the Papanicolaou (PAP) and the May-Grünwald-Giemsa (MGG) staining method for cytologic smears PAP wet fixation (ethanoi, methanol, etc.) cell shrinking nuclear details
MGG air-dried cell enlargement cytoplasmic details and extracellular substances
suited for comparison with histological sections (hematoxyUn)
3.1 FIXATION OF SMEARS
Papanicolaou staining. Smears are fixed immediately in 95% ethanol. Fixation in 95% methanol or in Delaunay solution*^ gives equally good results. Fixation must last at least 5 min and may be extended up to 24 h. For smears rich in blood, 3 % acetic acid may be added to the fixation medium to induce hemolysis of erythrocytes." May-Grünwald-Giemsa staining. Unfixed, air-dried smears (like hematological smears) are used. 3.2 STAINING
3.2.1 Papanicolaou (PAP) Staining Dip the ethanol-fixed smears into: — celloidin (0.25 Vo) in ether/ethanol (1:1) — ethanol 96% — ethanol 70% — ethanol 35 Ψο — distilled water — Harris hematoxylin (I), 5 min — tap water, rinse briefly — HCl(0.5%),30sec — tap water, 10 min, several changes — distilled water — ethanol 35% — ethanol 70% — ethanol 96% (2 X) — Orange OG 6, 5-7min (II) — ethanol 96% (2 X) — EA 50, 5 min (III) --ethanol 96% — ethanol abs., 1 min — ethanol abs., 2 min — xylene, 1 min — xylene, 2 min — mount in Eukitt
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(I) Harris hematoxylin solution: dissolve 1 g hematoxylin in 10 ml absolute ethanol. Dissolve 20 g aluminium potassium sulfate dodecahydrate (potassium alum) in 200 ml distilled water. Mix both solutions after 24 Κ heat to boiling point and add 0.5 g HgO, cool to room temperature and filter. Filter again before use. (II) and (III) Orange OG 6 and EA 50 solutions (Merck, Darmstadt, FRG) 3.2.2 May-Grünwald'Giemsa (MGG) Staining Put air-dried smears into: — May-Griinwaldsolution (I), 5 min — May-Grtinwaldsolution/distilled water 1:1, 5 min — put directly (without rinsing) into Giemsa solution (II), 20 min — rinse in distilled water — allow to dry in air — mount in Eukitt (I) May-Grünwald solution: dissolve\0.25 g methylene blue eosinate in 100 ml methanol under heating and filter (II) Giemsa stock solution (Merck, Darmstadt FRG)/distilled water 1:1 3.3CYTODL\GNOSIS
Only those parts of the smears containing clusters of hepatocytes in monolayers and without too much admixture of blood should be examined. In a PAP smear, hepatocytes are well preserved and appear as polygonal cells with distinct cell borders. Their cytoplasm is interspersed with many small, red granules and larger, cloudy basophilic particles. The nuclei are regular in size and structiu-e and contain one or more distinct nucleoli and a fine, regular chromatine pattern. Different size classes (diploid, tetraploid and larger nuclei) can be recognized. Many binuclear cells are present (Fig. 4). Apart from many blood cells, the smears frequently contain cholangioductular cells appearing as branched cords of small cells with tightly packed cuboid or elongated nuclei smaller than hepatocyte nuclei. These bile ductules can be readily identified when the smears are stained for y-glutamyl transpeptidase (p. 25). In contrast to normal hepatocytes, they show a marked enzyme activity. Kupffer cells of various size are identified by their ingested material. Sometimes a sheet of mesothelial cells from the peritoneum can be seen. In PAP smears, much attention must be given to the interpretation of nuclear size and chromatin pattern. It is important, therefore, not to over- or under-stain the nuclei. This precaution is essential since nuclear hyper- or hypo-chromasia are important indicators for the diagnosis of prenecrotic or neoplastic conditions. Rat Uver cytology has been described in more detail by Boelsterli and Zbinden.^®" For comparison, human liver FNAB cytology is likewise well documented.""•^^· 26. 31-34. 54. 55. 59
Examples. (1) Male ZUR:SIV-Z rats (100 g initial weight) were given the hepatocarcinogen, N-nitrosomorpholine (NNM) (10 mg/100 ml drinking water) for 116 days. Dysplastic hepatocytes and hepatocellular carcinoma cells (Fig. 5) were
Fine-needle Aspiration Biopsy of Rat Liver 23 identified in FNAB smears and correlated with histopathological studies in the same animals.^^ (2) Male ZUR:SIV'Z rats (250 g) were treated with the mitogen, butylated hydroxytoluene (BHT) (500 mg/kg by gastric intubation for 9 days). Liver hyper trophy and hyperplasia was readily recognized (Fig. 6). (3) Male ZUR:SIV-Z rats (250 g) were injected the necrogenic hepatotoxin, carbon tetrachloride (1 ml/kg i.p. in oil, 48 h prior to FNAB sampling). Necrotic hepatocytes (pycnotic nuclei, fatty infiltration) were a common finding in the smears (Fig. 7). 4. Cytochemistry Cytochemical reactions performed on cryostat or paraffin-embedded histological sections can be adapted for FNAB smears. Care must be taken with regard to the type and duration of fixation and the specificity of the reaction (negative and positive controls).^ 4.1 TRIGLYCERIDE STAINING FOR DEMONSTRATION OF HEPATIC STEATOSIS
Accumulation of triglycerides in the liver occurs frequently as a consequence of toxic, metaboUc or nutritional disturbances and is, therefore, a common and important finding in chronic toxicity studies. Lipid accumulation is readily recognized in smears after staining with Fettrot 7B (Fig. 8). It can be scored semiquantitatively.^® " ^ ^Fixa smear for 5 min in 4Vo formaline (CaCOybuffered), containing 1 % CaCl — rinse with distilled water — ethanol35Vo (dip) — ethanol 70% (dip) — Fettrot 7B solution (I), 15 min uptolh — ethanol 70% (dip) — distilled water — Harris hematoxylin (II), 2 min — tap water, 10 min (several changes) — distilled water — mount in glycerol jelly. Triglycerides stain red. (I) Fettrot 7B stock solution: 0.5 g Fettrot 7B (Chroma, Stuttgart, FRG) dissolved in 100 ml isopropanol. Working solution: stock solution (6parts) + distilled water (4 parts), stand for 24 h, then filter (II) as described for Papanicolaou staining (p. 21). Example. Hepatic steatosis was induced in male ZUR:SIV-Z rats (200 g) by feeding a high fat diet for 2-16 days.^^ Mild to severe triglyceride accumulation was readily identified and showed good correlation with histological evaluation of the same liver lobes. 4.2 GLYCOGEN STAINING FOR DEMONSTRATION OF HEPATIC GLYCOGENOSIS
Apart from hereditary or alimentary disturbance of glycogen metabolism, glycogen storage is a feature concomitant with the development of neoplastic nodules in rat
24
υ . BOELSTERLI AND G. ZBINDEN
liver during experimental hepatocarcinogenesis.^'^ It must be emphasized, however, that minor alterations cannot be recognized due to physiological fluctuations of glycogen content as a function of circadian rhythm, nutritional state, topographic localization within the hepatic lobule, etc. Glycogen can be stained cytochemically by the periodic acid-Schiff (PAS) method (Fig. 9). — Fixa smear in ethanol, 5 min up to 24 h — dip slide into a 2% solution of celloidin in ether/ethanol 1:1 — dry for a few sec in air — 70% ethanol, 2 min — periodate solution (I), 10 min -- tap water, 10 min (several changes) — distilled water — Schiff reagents (II), 20 min — NaHSO^solution (III), 3x1 min — tap water (20 min), several changes — Harris hematoxylin (IV), 2 min — tap water, 10 min (several changes) — distilled water — 35% ethanol — 70% ethanol — ethanol abs, — ether/ethanol 1:1 (celloidin coat removed) — xylene, 1 min — xylene, 2 min — mount in Eukitt. Glycogen stains red. (I) periodate solution: dissolve 0.8 g periodic acid and 0.25 g sodium acetate in 20 ml distilled water, add 80 ml ethanol abs. (II) Schiff reagents (Merck, Darmstadt, FRG) (III) NaHSO^ solution: 5 ml NaHSO^ (10%) + 5mlHCl(lM) + 100 ml distilled water. Always prepare a fresh solution (IV) Harris hematoxylin as described for Papanicolaou staining (p. 21) 4.3 IRON STAINING FOR DEMONSTRATION OF HEPATIC SIDEROSIS
Iron loading of hepatocytes and/or Kupffer cells is demonstrated by iron staining of FNAB smears.^^' ^· This has recently become important as a negative marker for preneoplastic and neoplastic hepatocytes in rat hepatocarcinogenesis. These altered cells do not accumulate iron after long pretreatment with an iron-rich diet." — — — — —
Use iron-free glassware (rinse with HCl) fix smears in 4% neutral formalin/96% ethanol (1:9) for 1 min rinse in distilled water let dry in air stain in potassium ferrocyanide (10% solution in distilled water) for 5 min (prepare freshly) — add 0.5 parts of 10% HCl, stain for 30 min — rinse in distilled water — let dry in air
FIGS. 4-11. For captions see overieat.
FIGS. 4-11. Cytological smears of rat liver obtained by FNAB. (see overleaf) FIG. 4. Normal hepatocytes (p. 22 ). PAP staining. Regular polygonal cells with distinct cell borders and typical cytoplasmic staining with red granulation and cloudy basophilic particles. Regular nuclei with one or more distinct nucleoh. Note many binuclear hepatocytes. χ 400.
FIG. 5. Hepatocytes of a rat treated with iV-nitrosomorpholine (10 mg/100 ml in drinking water) for 116 days (p. 22 ). Apart from neoplastic nodules, hepatocellular carcinoma has developed. PAP staining. Irregular, basophilic cells featuring marked anisokaryosis and nuclear crowding. The hypochromatic nuclei harbour one or more distinct nucleoli of various size and shape, χ 400.
FIG. 6. Hepatocytes of a rat treated with butylated hydroxytoluene (500 mg(kg p.o. for 9 days) (p. 23 ). PAP staining. The hypertrophic hepatocytes are swollen and show huge polyploid nuclei with the typical chromatin pattern. The red cytoplasmic granulation which is present in normal hepatocytes is missing, χ 400.
FIG. 7. Hepatocytes of a rat treated with carbon tetrachloride (1 ml/kg i.p. in oil, 48 h prior to FNAB) (p. 23). PAP staining. A group of necrotic hepatocytes featuring cytoplasmic vacuolization, nuclear pycnosis, nuclear marginal chromatin condensation and hyperchromasia. χ 400.
FIG. 8. Liver cells of a rat fed a high-fat diet (p. 23) for 2 weeks. Severe hepatic steatosis has developed. Triglyceride droplets in hepatocytes are stained intensely red with Fettrot 7B X 310.
FIG. 9. Hepatocytes of a rat treated with 7V-nitrosomorpholine (10 mg/100 ml drinking water) for 84 days. Altered hepatocytes within neoplastic nodules are characterized by marked glycogen storage which is stained purple with the PAS reaction (p. 24 ). Note intranuclear glycogen, χ 400.
FIG. 10. Liver cells of a rat treated with N-nitrosomorphohne (10 mg/100 ml drinking water) for 28 days stained for y-glutamyl transpeptidase (7-GT) activity (p. 25). Apart from the cholangioductular cells (branched cords) which exhibit a marked 7-GT activity, the carcinogen-altered hepatocytes of preneoplastic foci or within the neoplastic nodules show a positive γ-GT reaction outlining the cell membranes, χ 250.
FIG. 11. Hepatocytes of a rat treated with 7V-nitrosomorpholine (10 mg/100 ml drinking water) for 70 days stained for glucose-6-phosphatase (G-6-Pase) activity (p. 26). In contrast to normal hepatocytes which show a diffuse cytochemical reaction in the cytoplasm but not in the nucleus, these carcinogen-altered cells display a focal intranuclear enzyme activity and in some cells only a weak or missing cytoplasmic G-6-Pase activity, χ 250.
Fine-needle Aspiration Biopsy of Rat Liver counterstain with 1% neutral red, 3 min rinse with distilled water and let dry mount in Eukitt. Iron stains blue.
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4.4 GLUTAMYL TRANSPEPTIDASE STAINING FOR DEMONSTRATION OF PRENEOPLASTIC AND NEOPLASTIC HEPATOCYTES
Evidence is accumulating that a manyfold rise in 7-glutamyl transpeptidase (γ-GT) activity in rat hepatocytes during experimental hepatocarcinogenesis is an early and reUable marker for carcinogen-altered liver cells in preneoplastic foci, neoplastic nodules and hepatocellular carcinoma." ^* " 2® A positive enzyme reaction is observed in fetal and neonatal hepatocytes but not in normal adult rat liver.^ After exposure to a carcinogen hepatocytes featuring a positive enzyme reaction outhning the plasma membranes are readily identified (Fig. 10). Normal hepatocytes give a negative or only very faint color reaction. In all livers, cholangioductular cells display a marked y-GT activity (p. 22). The histochemical method of Albert et aU with y-glutamyl-a-naphthylamide as substrate is used. The liberated y-glutamyl moiety is accepted by glycylglycine, and the a-naphthylamine is coupled to Fast Garnet GBC, a diazonium salt, to form an insoluble red precipitate of diazo dye. Wash FNAB samples free from blood in ice-cold PBS (p. 20), transfer them onto a glass slide with a drop of PBS and prepare a smear. Fix immediately in ice-cold absolute ethanol for 10 min. Let dry in air. Slides can be stored at 4''C. — Encircle the fixed, air-dried cells on the glass slide with a fat pencil, place a few drops of substrate solution (III) on them and incubate for 30 min at 25 °C — rinse in 0.9% NaCl, 3x1 min — CuSO^(0.1M),2min — distilled water — Harris hematoxylin (IV), 1-2 min — tap water, 10 min (several changes) — distilled water — mount in glycerol jelly (I)
200 μmoles y-glutamyl-a-naphthylamide (= 5.45 mg) (Serva, Heidelberg, FRG) dissolved in 60 ml NaCl (0.9%) (II) 1 mmol glycylglycine (= 132 mg) dissolved in 40 ml phosphate buffer (0.1 M, pH6.7) (III) immediately before use, mix (I) and (II) and add 50 mg Fast Garnet GBC (Serva, Heidelberg, FRG), stir, filter (IV) Harris hematoxylin as described for Papanicolaou staining (p. 21). Example. Male ZUR.SIV-Z rats (170 g initial weight) were given the hepatocarcinogen, N-nitrosomorpholine (NNM) (10 mg/100 ml drinking water) for 56 days. Already after 1 week of treatment, about 50% of the animals were found to contain y-GT positive hepatocytes in FNAB smears, and after 4 weeks of treatment, almost all of the smears searched were positive." (See Fig. 10.)
26 υ . BOELSTERLI AND G. ZBINDEN 4.5 AMINO ACID NAPHTHYLAMIDASE STAINING FOR DEMONSTRATION OF BILE CANALICULI IN CHOLESTASIS
The cytochemical staining method for FNAB smears in human liver and that of rat and guinea pig is described in detail by Wasastjerna.^^ ^® 4.6 ALKALINE PHOSPHATASE STAINING
Increased alkaline phosphatase activity is, according to Ekelund,^^ a highly sensitive method for detecting mild bile stasis in smears. The cytochemical method for FNAB smears is described by Wasastjerna.^* 4.7 ADENOSINE TRIPHOSPHATASE (ATPase) AND GLUCOSE-6-PHOSPHATASE (G-6-Pase) STAINING
Focal loss of ATPase and G-6-Pase have been reported to occur in preneoplastic foci and neoplastic nodules of rat liver treated with various hepatocarcinogens.^^ *® The canalicular ATPase and the cytoplasmic G-6-Pase can easily be demonstrated by cytochemical methods (Fig. 11). However, as mentioned above, care must be taken in interpreting the results, as enzyme activity fluctuates in the rat Uver as to the site of the cells investigated within a hepatic lobule. The method of Wachstein and MeiseP® are appUed as modified by Rabes et al.*^ 4JA
ATPase — Wash FNAB samples in ice-cold sucrose/EDTA (0.25 M/O.OOl M), transfer them onto glass slides, prepare a smear, let dry — fix with glutaraldehyde (2.5Ψο in cacodylate buffer, p. 27) for 5 min. Rinse with buffer solution. Let dry. — encircle the fixed, air-dried cells on the glass slide with a fat pencil, place a few drops of substrate solution (I) on them and incubate for 15 min at37''C — rinse with distilled water — ammonium sulfide (1 % aqueous solution), 1 min — rinse with distilled water — mount in glycerol jelly. Site of ATPase activity stains brown-black (lead sulfide) (I) substrate-buffer solution: tris-maleate buffer (0.25 M, pH 7.2), MgSO^. 7H^O (5mM), Pb(NO^)2 (5mM), ATP.Na2(0.8 mM).
4.7.2 G-6-Pase Use the same procedure as for A TPase, except: — use unfixed smears — use glucose-6-phosphate.Na2 (0.8 mM) as substrate instead of A TP.Nai — after incubation with ammonium sulfide and rinsing with distilled water, postfix the smear in neutral 4Vo formalin for 5 min. (SeeFig. 11.)
Fine-needle Aspiration Biopsy of Rat Liver
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5 Special Techniques 5.1 ELECTRON MICROSCOPY
Liver FNAB samples can be processed for electron microscopy by the usual technique.^2 Since the material often consists of very smaU fragments, rinsing and changing fixatives must be performed with great caution. The influence of the duration of glutaraldehyde fixation is evaluated by Hagelqvist" using FNAB samples of guinea pig hver and salivary gland. The penetration of the fixative into the tissue of needle biopsies from human Uver is studied by Petersen.*^ Expel the aspirated material in needle and syringe directly into a glutaraldehyde solution (ice-cold) (I), wash in cacodylate buffer (II), postfix in osmium tetroxide (III), dehydrate with dimethoxypropane^^ and embed in an epoxy resin as for routine electron microscopy. (I)
glutaraldehyde solution: 10 ml glutaraldehyde (25%), 50 ml cacodylate buffer (II), 30 ml bidistilled water, adjust to pH 7.3 and fill up with water to 100 ml (II) cacodylate buffer: 0.2 Af sodium cacodylate, pH 7.3 (III) 2% O5O4 in cacodylate buffer
5.2 FLUORESCENCE MICROSCOPY AND POLARIZATION MICROSCOPY FOR DEMONSTRATION OF DRUG-INDUCED PHOSPHOLIPIDOSIS
PhosphoUpidosis induced by amphiphilic drugs^' manifests itself as storage of myelin bodies in the lysosomes of hepatocytes. These can be recognized either in the polariza tion microscope, appearmg as *'Maltese cross" bodies (birefringent figures)" or, if the drug itself is a fluorochrome (as in the case of mepacrine) by fluorescence micro scopy."Wash FNAB samples free from blood in ice-cold PBS (p. 20), transfer them onto a glass slide with a drop of PBS and prepare a smear. Cover immediately with a coverslip and surround with nail varnish. Examine the slides in polarized light (oil immersion, 100 x objective) or in the fluorescence microscope (filter combina tion as for acridine orange fluorescence). Example. Male rats of the ZUR:SIV-Z strain (250 g) were treated with mepacrine (Geistlich, Wolhusen, Switzerland) (100, 200, 400 and 800 mg/kg respectively by gastric intubation for 2 days), or with chlorphentermine (CFS, Basel, Switzerland) (50 mg/kg by gastric intubation for 10 and 35 days respectively). A control group received water. The results were graded semiquantitatively and are Ipresented in Table II (0 no detectable fluorescent bodies/Maltese cross bodies; 1 + few and small bodies; 2 + small and large bodies in many cells; 3 + many bodies in all cells). The results indicate that the cellular inclusions were recognized in all smears and verified as myelin bodies (lysosomes) by electron microscopical examination (p. 27) of FNAB samples of the same animals. 5.3 ENZYMATIC DISSOCIATION AND CYTOFLUOROMETRY
Liver FNAB samples consist of smaU tissue cylinders of 1-2 mm up to maximaUy 10 mm in length. Apart from these grossly visible fragments, many small clusters of hepatocytes and even single cells are also aspirated and are especially abundant in
28
υ . BOELSTERLI AND G. ZBINDEN TABLE II. Semiquantitative evaluation of mepacrine- and chlorphentermine-induced myelin bodies in rat hepatocytes obtained by FNAB and investigated in cytological smears by fluorescence microscopy (FM) and polarization microscopy (PM) (Explanation of grading in the text, p. 27). control
FM PM
chloφhentermine (days treated with 50 mg/kg/d)
mepacrine dose (mg/kg/d for 2 days) 100
200
400
800
1+ 1+
1+ 1+
2+ 2+
3-f 3+
10
1+
35
1+
liver samples from rats treated with various hepatotoxic agents. Usually these fragments are smeared on glass slides for microscopical examinations. Cytofluorometric studies on smears, however, proved to be of minor value as the cells are not always present in monolayers and are intermingled with various blood cells. Thus, enzymatic dissociation of the FNAB specimens and further studies on cell suspensions should be preferred for cytofluorometric investigations of DNA,® enzyme activity (γ-GT)^^ or other assays. The effect of the negative pressure during aspiration biopsy on the viability of single cells was evaluated by Plesnicar et al.**^ 6 Biochemical Assays In addition to cytochemical studies (p. 23), enzyme activities can also be determined quantitatively in the liver FNAB homogenate. However, the activity cannot be ascribed to a certain cellular type, and the origin of the homogenized sample as to the location within the liver lobe remains obscure. Although the enzyme pattern is heterogeneous within the liver lobule (reviewed by Jungermann and Sasse^O good results have been obtained in FNAB studies of normal and pathological livers in man^^-^^ *® and in rodents.^ " In toxicity studies, enzymes involved in biotransformations (detoxification and activation) of xenobiotics are especially important and are described and discussed in detail later in this book. Of great value is the possibility of assaying several enzymes in the same homogenate. In order to express a specific enzyme activity, the measured activity should be related to the protein content of the homogenate. Literature 1. ALBERT, Z., J. ORLOWSKA. M. ORLOWSKI and A. SZEWCZUK (1964). Histochemical and biochemical investigations of gamma-glutamyl transpeptidase in the tissues of man and laboratory rodents. Acta Histochem. 18,78-89. 2. ALBERT, Z., Z. RZUCIDLO and H. STARZYK (1970). Comparative biochemical and histochemical studies on the activity of gamma-glutamyl transpeptidase in the organs of fetuses, newborns and adult rats. ActaHistochem, 37,34-39. 3. BANNASCH, P. (1968). The Cytoplasm of Hepatocytes during Carcinogenesis, Recent Results in Cancer Research, Vol. 19. Springer, Berlin, Heidelberg, New York. 4. BANNASCH, P. (1978). Cellular and subcellular pathology of liver carcinogenesis. Primary Liver Tumors (Edited by H. Remmer et al.), MTP Press, Lancaster, pp. 87-111. 5. BANNASCH, P. and H. ANGERER (1974). Glykogen und Glukose-6-Phosphatase wfthrend der Kanzerisierung der Rattenleber durch N-nitrosomorpholin. Arch, Geschwulstforsch, 43, 105-114. 6. BAUNSGAARD, P., G. C. SANCHEZ and C. J. LUNDBORG (1979). The variation of patiiological changes in the liver evaluated by double biopsies. Acta Path, Microbiol. Scand. A 87,51-57.
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7. BERNE, C. and S. WESTMAN-NAESER (1978). Enzymatic studies of uver ceUs obtained by fineneedle biopsy from mice with the obese-hyperglycemic syndrome (genesymbol ob). Horm, Metab, Res. 10.124-127. 8. BICHEL, P., P. FREDERIKSEN, T. KJAER, P. THOMMESEN and L. L. VINDELÓV (1977). Flow microfluorometry and transrectal fine-needle biopsy in the classification of human prostatic carcinoma. Cancer Añ, 1206-1211. 9. BOELSTERLI, U. (1979). Gamma-glutamyl transpeptidase—an early marker for hepatocarcinogens in rats. Trends in Pharmacological Sciences 1, 47-49. 10. BOELSTERLI, U. and G. ZBINDEN (1978). The use of fine-needle aspiration biopsy for the diagnosis of hepatic steatosis in rats. Toxicology Letters 2,35-43. 11. BOELSTERLI, U. and G. ZBINDEN (1979). Application of fine-needle aspiration biopsy for the diagnosis of dysplastic and neoplastic liver cell changes induced by N-nitrosomorpholine in rats. Arch. Toxicol. Al, 225-238. 12. BOLL, I. (1969). Das Zytohepatogramm. Fortschr. Med. 87,420-422. 13. BRITS, C. J. (1974). Liver aspiration cytology. South Afr. Med. J. 48,2207-2214. 14. CAMERON, R., J. KELLEN, A. KOLIN, A. MALKIN and E. FARBER (1978). γ-Glutamyltransferase in putative premalignant liver cell populations during hepatocarcinogenesis. Cancer Res. 38, 823-829. 15. CARNEY, C. N. (1975). CUnical cytology of the liver. Acta Cytol. 19,244-250. 16. DANNMEIER, H. (1973). Die Feinnadelbiopsie der Leber. Verh. Dtsch. Ges. Path. 57, 243-246. 17. DOMINIS, M., S. CERLEK and D. SOLTER (1973). Cytology of diffuse Uver disorders. Acta Cytol. 17,205-208. 18. DULBECCO, R. and M. VOGT (1954). Plaque formation and isolation of pure lines with poliomyelitis viruses. / . Exp. Med. 99,167-182. 19. EKELUND, P. (1971). A cytological study of extra- and intrahepatic cholestasis in the rat. Ann. Med. Exp. Biol. Fenn. 49,49-53. 20. ELEQUIN, F. T., F. M. MUGGL\, N. A. GHOSSEIN and K. SCHREIBER (1977). A quick method for concentrating and processing cancer cells from serous fluids and fine-needle aspiration aspirates. Acta Cytol. 596-599. 21. G O S S N E R , W . and H. F R I E D R I C H - F R E K S A (1964). Histochemische Untersuchungen über die Glukose-6-Phosphatase in der Rattenleber wáhrend der Cancerisierung durch Nitrosamine. Z. Naturforsch. 19,862-863. 22. HAGELQVIST, E. (1978). Light and electron microscopic studies on material obtained by fine-needle biopsy. Acta Otolaryngol., Suppl. 354. 23. HANSEN, H. A. and A. WEINFELD (1959). Hemosiderin estimation and sideroblast counts in the differential diagnosis of iron deficiency and other anemias. Acta Med. Scand. 165,333-357. 24. HARADA, M., K. OKABE, K. SHIBATA, H. MASUDA, K. MIYATA and F. ENOMOTO (1976). Histochemical demonstration of increased activity of γ-glutamyl transpeptidase in rat liver during hepatocarcinogenesis. Acta Histochem. Cytochem. 9,168-179. 25. HENRIQUES, U. V. and K. HASSELSTRÓM (1977). Evaluation of jaundice: fine-needle aspiration hver cytology as a discriminating tool. Dan. Med. Bull. 24,104-108. 26. JOHANSEN, S. and J. MYREN (1971). Fine-needle aspiration biopsy smears m the diagnosis of liver diseases. Scand. J. Gastroenterol. 6,583-588. 27. JUNGERMAN, K. and D. SASSE (1978). Heterogeneity of Uver parenchymal cells. Trends in Biochemical Sciences 198-202. 28. KALENGAYI, M. M. R., G. RONCHI and V. J. DESMET (1975). Histochemistry of gamma-glutamyl transpeptidase in rat liver during aflatoxin B^-induced carcinogenesis. / . Natl. Cancer Inst. 55, 579-588. 29. LÜLLMANN, H., R. LÜLLMANN-RAUCH and O. WASSERMANN (1975). Drug-induced phospholiposes. Crit. Rev. Toxicol. 4,185-218. 30. LUNDIN, P., A. LUNDQUIST and O. LUNDVALL (1969). Evaluation of fine-needle aspiration biopsy smears in the diagnosis of liver iron overload. Acta Med. Scand. 186,369-373. 31. LUNDQUIST, A. (1970). Fine-needle aspiration biopsy for cytodiagnosis of malignant tumour in tiie liver. Acta Med. Scand. 188,465-470. 32. LUNDQUIST, A. (1970). Liver biopsy with a needle of 0.7 mm outer diameter. Acta Med. Scand. 188,471-474. 33. LUNDQUIST, A. (1971). Fine-needle aspiration biopsy of Üie Uver. Acta Med. Scand., Suppl. 520. 34. LUNDQUIST, A. and M. AÍKERMAN (1970). Fine-needle aspiration biopsy in acute hepatitis and liver cirrhosis. Ann. Clin. Res. 2,197-203. 35. LUNDQUIST, A. and P. OCKERMAN (1969). Fine-needle aspiration biopsy of tiie Uver in healtiiy adults. Activity of lysosomal acid hydrolases. Enzym. Biol. Clin. 10,300-304. 36. LUNDQUIST, A. and P. OCKERMAN (1970). Fine-needle aspiration biopsy of human Uver for enzymatic diagnosis of glycogen storage disease and gargoyUsm. Acta Med. Scand. 59, 293-2%.
30 υ . BOELSTERLI AND G. ZBINDEN 37. LUNDQUIST, Α., P. A. OCKERMAN and B. SCHERSTEN (1969). Fine-needle aspiration biopsy of the Uver in healthy adults. Activity of enzymes of glycogen utilization and glucose production. Enzym. BioL 10.8-12. 38. MENGHINI, G. (1970). One-second biopsy of the liver. New J, Med, 283, 582-585. 39. MULLER, L. L. and T. J. JACKS (1975). Rapid chemical dehydration of samples for electron micro scopic examination. / . Histochem Cytochem, 23,107-110. 40. OCKERMAN, P. A. (1%7). Glucose-6-phosphatase assay on microgram amounts of liver tissue. Clin, Chim, Acta 17,201-206. 41. OCKERMAN, P. A. (1968). A technique for the enzymatic diagnosis of glycogen storage disease on very small tissue specimens. Acta Paediat. Scand, 57,105-109. 42. PEARSE, A. G. E. (1968-1972). Histochemistry, Churchill Livingstone, Edinburgh, London. 43. PEDIO, G. (1976). Diagnose und Schnelldiagnose der Brustdrüsentumoren durch Feinnadelpunktion. Schweiz, Med, Wschr, 106,477-480. 44. PETERS, T. J. and C. A. SEYMOUR (1978). Analytical subcellular fractionation of needle-biopsy specimens from human liver. Biochem, J, 174,435-446. 45. PETERSEN, P. (1977). Glutaraldehyde fixation for electron microscopy of needle biopsies from human livers. Acta Path, Microbiol, Scand, A 85,373-383. 46. PLESNICAR, S., C. RUBIO, A. SIGURDSON and J. ZAJICEK (1968). Studies on the effects of aspiration biopsy on aspirated cells. Acta Cytol. 12,454-461. 47. RABES, H., P. SCHOLZE and B. JANTSCH (1972). Growth kinetics of diethyl-nitrosamine-induced enzyme-deficient ^'preneoplastic** liver cell populations in vivo and in vitro. Cancer Res, 32, 2577-2586. 48. SCHAUER, A. and E. KUNZE (1968). Enzymhistochemische und autoradiographische Untersuchungen wáhrend der Cancerisierung der Rattenleber mit Diáthyhiitrosamin. Ζ. Krebsforsch, 70, 252-266. 49. SCHAUER, A. and E. KUNZE (1976). Tumours of the Uver, Pathology of Tumours in Laboratory Animals, vol. 1, Tumours of the Rat, part 2 (Edited by V. S. TURUSOV), L\RC Sci. Publ. 6, Lyon, pp. 41-72. 50. SÓDERSTRÓM, Ν. (1966). Fine-needle aspiration biopsy, Ahnqvist & WikseU, Stockholm. 51. SOLOWAY, R. D., A. H. BAGGENSTOSS, L. J. SCHOENFIELD and W. H. J. SUMMERSKILL (1971). Observer error and sampling variability tested in evaluation of hepatitis and cirrhosis by liver biopsy. Am, J, Dig, Dis, 16,1082-1086. 52. SOOST, H. J. (1976) Lehrbuch derKlinischen Zytodiagnostik, Georg Thieme, Stuttgart. 53. STAIGER, G. R. (1974). Light-microscopic demonstration of drug-induced myelin bodies in the liver of the rat. Experientia 30,385-386. 54. STORMBY, N. and M. AKERMAN (1973). Aspiration cytology in the diagnosis of granulomatous Uver lesions. Acta Cytol, 17,200-204. 55. SZASZ, G., L. SAFRANY and A. LUX (1%9). Die Zytologie des Leberpunktats. Dtsch, Gesundheitswes, 22,1306-1311. 56. TATEMATSU, M., T. SHIRAI, H. TSUDA, Y. MIYATA, Y. SHINOHARA and N. ITO (1977). Rapid production of hyperplastic liver nodules in rats treated with carcinogenic chemicals: a new approach for an in vivo short-term screening test for hepatocarcinogens. GANN6$, 499-507. 57. VANDERLAAN, M., C. CUTTER and F. DOLBEARE (1979). How microfluorometric identification of Uver cells with elevated ganmia-glutamyltranspeptidase activity after carcmogen exposure. J. Histochem, Cytochem, 27,114-119. 58. WACHSTEIN, M. and E. MEISEL (1957). Histochemistry of hepatic phosphatase at a physiologic pH with special reference to the demonstration of bile canaliculi. Amer, J, Clin, Pathol, 27, 13-23. 59. WASASTJERNA, C. (1979). Liver, Aspiration Biopsy Cytology, part 2, Cytology of Infradiaphragmatic Organs (Edited by J. ZAJICEK) Karger, Basel, München, Paris, London, New York, Sydney. 60. WASASTJERNA, C. and P. EKELUND (1974). The amino add naphtiiylamidase reaction of the bile canaliculi in Uver smears. Acta Cytol, 18,23-29. 61. WASASTJERNA, C , P. REISSELL, J. KARJALAINEN and P. EKELUND (1972). Fatty Uver in diabetes. A cytological study. Acta Med, Scand, 191,225-228. 62. WILLL\MS, G. M. and R. S. YAMAMOTO (1972). Absence of stainable iron from preneoplastic and neoplastic lesions in rat Uver with 8-hydroxyquinoUne-induced siderosis. J, Natl, Cancer Inst, 49,685-692. 63. WILLIAMS, G. M., M. KLAIBER. S. E. PARKER and E. FARBER (1976). Nature of early appearing, carcinogen-induced Uver lesions resistant to iron accumulation. / . Natl, Cancer Inst, 57, 157-165. 64. WITTEKIND, D. and J. STAUBESAND (1970). Ueber farbstoffabhángige Variationen zeUularer Autophagie. I. Mitteilung: Neutrakot und Mepacrin. Cytobiologie 2,275-314. 65. ZAJICEK, J. (1974). Aspiration Biopsy Cytology, part I. Cytology of Supradiaphragmatic Organs. Karger, Basel. 66. ZAJICEK, J. (1977). Aspiration biopsy cytology. Lymphology 10,94-101.
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67. ZAJICEK. J. (1979). Aspiration Biopsy Cytology, part 2. Cytology of Infradiaphragmatic Organs. Karger, Basel, München, Paris, London, New York, Sydney. 68. ZAJICEK, J., T. CASPERSSON, P. JAKOBSSON, J. KUDNYNOWSKI, J. LINSK and M. US-KRASOVEC (1970). Cytologic diagnosis of mammary tumors from aspiration biopsy smears. Comparison of cytologic and histologic findings in 2,111 lesions and diagnostic use of cytophotometry. Acta Cytol. 14, 370-376. 69. ZETTERBERG, A. and P. L. ESPOSTI (1976). Cytophotometric DNA-analysis of aspirated cells from prostatic carcinoma. Acta Cytol. 20,46-57.
FINE-NEEDLE ASPIRATION BIOPSY OF RAT LIVER: TECHNIQUES AND APPLICATIONS U. BOELSTERLI AND G. ZBINDEN
1 Introduction It is now generally accepted that the fine-needle aspiration biopsy (FNAB) method which is widely used in clinical medicine yields reliable and reproducible results in the cytodiagnosis of human tumors (chapter 2 in this book). The complication rate of the procedure is low. Thus, there are very few contraindications. Furthermore, the biopsy technique is simple and rapid, and can be performed repeatedly.*®Considering these favorable cUnical experiences, it was logical to try to adapt this method for cytological investigations in small laboratory animals.^® In experimental toxicology, the objectives of the studies differ to some extent from those in clinical pathology. The clinician is primarily interested in the diagnosis of tumors and in assessing their dignity. In experimental studies the aim of the method is to recognize in the living animal a variety of pathological alterations which can be caused by chenucal substances. Although FNAB sampling can not always replace conventional histology, the advantages of the method are obvious: first, it is not necessary to sacrifice an animal just to get a small piece of tissue. An even more important advantage is the possibility of monitoring individual animals by serial punctures over a long period of time. In this way, individual biological variations can largely be eliminated. A biopsy is, of course, not always representative of the whole organ. A certain sampling error must therefore be taken into consideration.*^ The importance of this error varies according to the nature of the lesion: a diffuse lesion is much more likely to be identified than one which is focal. In order to increase sampling accuracy, it is recommended to take biopsies from several sites of the organ and to perform two FNAB at the same time.* In small laboratory animals, an organ which is to be punctured successfully has to meet the following requirements: it must be within easy reach for blind puncture or should be accessible to palpation. Furthermore, it should represent a relevant target for toxic effects. From this it is clear that the Hver was the first organ for which the FNAB technique was developed. 2 Uver FNAB in the Rat The rat liver is composed of five lobes: the right medial ( = right accessory) lobe, the left medial ( = left accessory) lobe, the right lateral lobe, the left lateral lobe, and the caudate lobe ( = papillary process of the right lateral lobe).*^ In order to avoid aspiration of gastric content, biopsies are taken from the right side of the liver. Care must be taken to puncture always the same lobes, i.e. the right medial and right lateral 17
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υ . BOELSTERLI AND G. ZBINDEN
liver lobes (Fig. 1). Since the rat lacks a gall bladder the risk of bile peritonitis is minimal. Routinely two punctures should be performed at the same time. Long-term experiments showed that even repeated liver FNAB did not affect hepatic structure and function." No deaths due to the punctures have, so far, been recorded. The slight bleeding into the peritoneal cavity that occurs after a puncture is of no consequence. The biopsy channel in the liver is replaced by fibrotic tissue containing a few macro phages with hemosiderin inclusions.
FIG. 1. Site of puncture in the rat liver. The xiphoid process (px) of the sternum is palpated, and the needle is inserted into the right medial liver lobe (rm) and the right lateral Uver lobe (rl). The needle should be in an angle of approx. 45** to the body surface, and attention should be paid to not penetrating too deeply with the needle nor to pierce the diaphragm (d). For details see text (p. 19).
2.1 ANESTHESIA AND PRELIMINARY TREATMENT
Anesthesia. The rat is given a slight ether narcosis and is placed with the dorsal side on an operating table where its limbs are slightly stretched and fixed (Fig. 2). During further manipulations, anesthesia is maintained by supplementary ether inhalation. So far no change in hepatocyte morphology or changes in the enzyme activity pattern have been recorded due to the very short and slight ether narcosis (c. 30 sec). Preliminary treatment is not necessary. There is no need to shave the ventral side of the rat. Since further processing of the aspirated material must be done very quickly, it is important that all necessary equipment (glass slides, fixatives) are in easy reach. 2.2 TECHNIQUE OF LIVER FNAB
Equipment. A handle for disposable syringes (Cameco, M. Bieri, CH-3097 LiebefeldBern, Switzerland), 20-ml plastic syringes (Luer type) and 22-gauge needles (0.7 mm in diameter) are necessary. The syringes are not filled with buffer solution and should be absolutely dry.
Fine-needle Aspiration Biopsy of Rat Liver 19 Technique (Figs. 1 and 2). The xiphoid process of the sternum is palpated. The needle is inserted at an angle of approx. 45° at the caudal right side of the xiphoid process. Deep penetration must be avoided. When the needle has entered the right medial and lateral liver lobes, the piston of the syringe is retracted and, while maintaining negative pressure, the needle is moved back and forth 2-3 times by about 1 cm. In this phase the needle is directed into different areas of the lobes. The piston is then released and the aspiration is interrupted. The needle is quickly retracted. It is disconnected from the syringe. The syringe is filled with some air and reconnected to the needle. The aspirated material is expelled immediately either on a glass sUde to prepare a smear or into a buffer or fixative solution (p. 20).
FIG. 2. Fine-needle aspiration biopsy of rat liver. The animal is in slight ether narcosis and fixed on an operating table. The biopsy is performed percutaneously with a fme needle (0.7 mm in diameter), attached to a 20-ml syringe. Puncture and aspiration is facilitated by using a special handle. The other hand thereby is free for palpating.
2.3 PROCESSING OF THE BIOPSY SPECIMEN
In contrast to conventional liver biopsy with the needle described by Menghini^' where a cylinder of hver tissue is obtained which can be fixed and further processed for histological studies, the FNAB technique yields a number of large and small tissue fragments. By one puncture one usually obtains 5-10 mg liver tissue which is sufficient for many quantitative biochemical assays. During aspiration, a varying amount of blood is admixed. The biopsy specimens can be treated by two different ways: (1) they are smeared on glass sUdes, fixed and stained with routine cytological methods or undergo cytochemical staining reactions for microscopic evaluation; or (2) they are washed free from blood in buffer solutions and collected either for homogenization for quantitative biochemical assay or undergo special treatment for fixation or enzymatic dissociation (p. 27).
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υ . BOELSTERLI AND G. ZBINDEN
2.3.1 Preparation of Smears For these preparations care should be taken not to aspirate too much blood. The material in the needle is expelled onto one or several glass sUdes by depositing a single drop. Usually the tip of the syringe contains valuable material which can also be blown out onto further sudes. A smear is prepared immediately, using a second glass slide (Fig. 3). It is essential for good staining quality that the preparation of the smear and fixation is performed before the blood has coagulated.
FIG. 3. Technique of smear preparation: (a) a single drop of the content in the needle or in the tip of the syringe is deposited on a clean glass slide; (b) it is immediately covered with a second glass slide, and with a slight press a smear is quickly prepared; (c) clusters of hepatic tissue are spread over the slide, intermingled with blood. Monolayers of hepatocytes should be preferred for microscopical examination.
2.3.2 Washing of the Samples In order to wash the biopsy samples free from blood (which is usually admixed during aspiration), the content of the needle and tip of the syringe is expelled into icecold phosphate-buffered saline (PBS)^^(I). Specimens are collected by means of a small syringe and subsequently washed by repeated transfers into fresh PBS. Other methods^" are also described to concentrate and process aspirated cells from tumors (FicoU gradient and cytocentrifuge). (7) PBS: 8.00 g NaCl, 0.20 g KCl, 1.15 g Na^PO^ 11 water (pH 7.4).
0.20 g KH^O^
dissolved in
2.3.3 Homogenization The biopsy samples are transferred into a microhomogenizer glass tube (Duall, 0.3-1.0 ml, Kontex, Vineland, NJ, USA) and homogenized in 0.3-0.6 ml PBS (ice-cold) with a teflon pestle either by 10 strokes (1500 rpm) or, even better, by manual rotating up-and-down movements (on ice). Enzyme assays are usually performed on the crude homogenate, since differential centrifugation of the homogenate would be very dif ficult. The homogenate is stirred each time before aliquots are taken. However, crude liver homogenates obtained by FNAB in mice were compared with freeze-dried samples which were shown to yield higher activities for some enzymes. ^ Moreover, a method for subcellular fractionation of biopsy specimens by density gradients was recently describ ed.^ 3 Cytology For cytological studies of FNAB smears, two staining techniques are routinely used: (1) Papanicolaou staining, and (2) May-Grünwald-Gíemsa staining." The two methods are compared in Table 1. In our laboratory, we almost exclusively use the Papanicolaou (PAP) staining for liver cytology which gives more representative and reliable results.
Fine-needle Aspiration Biopsy of Rat Liver
21
TABLE L Comparison of the Papanicolaou (PAP) and the May-Grünwald-Giemsa (MGG) staining method for cytologic smears PAP wet fixation (ethanoi, methanol, etc.) cell shrinking nuclear details
MGG air-dried cell enlargement cytoplasmic details and extracellular substances
suited for comparison with histological sections (hematoxyUn)
3.1 FIXATION OF SMEARS
Papanicolaou staining. Smears are fixed immediately in 95% ethanol. Fixation in 95% methanol or in Delaunay solution*^ gives equally good results. Fixation must last at least 5 min and may be extended up to 24 h. For smears rich in blood, 3 % acetic acid may be added to the fixation medium to induce hemolysis of erythrocytes." May-Grünwald-Giemsa staining. Unfixed, air-dried smears (like hematological smears) are used. 3.2 STAINING
3.2.1 Papanicolaou (PAP) Staining Dip the ethanol-fixed smears into: — celloidin (0.25 Vo) in ether/ethanol (1:1) — ethanol 96% — ethanol 70% — ethanol 35 Ψο — distilled water — Harris hematoxylin (I), 5 min — tap water, rinse briefly — HCl(0.5%),30sec — tap water, 10 min, several changes — distilled water — ethanol 35% — ethanol 70% — ethanol 96% (2 X) — Orange OG 6, 5-7min (II) — ethanol 96% (2 X) — EA 50, 5 min (III) --ethanol 96% — ethanol abs., 1 min — ethanol abs., 2 min — xylene, 1 min — xylene, 2 min — mount in Eukitt
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(I) Harris hematoxylin solution: dissolve 1 g hematoxylin in 10 ml absolute ethanol. Dissolve 20 g aluminium potassium sulfate dodecahydrate (potassium alum) in 200 ml distilled water. Mix both solutions after 24 Κ heat to boiling point and add 0.5 g HgO, cool to room temperature and filter. Filter again before use. (II) and (III) Orange OG 6 and EA 50 solutions (Merck, Darmstadt, FRG) 3.2.2 May-Grünwald'Giemsa (MGG) Staining Put air-dried smears into: — May-Griinwaldsolution (I), 5 min — May-Grtinwaldsolution/distilled water 1:1, 5 min — put directly (without rinsing) into Giemsa solution (II), 20 min — rinse in distilled water — allow to dry in air — mount in Eukitt (I) May-Grünwald solution: dissolve\0.25 g methylene blue eosinate in 100 ml methanol under heating and filter (II) Giemsa stock solution (Merck, Darmstadt FRG)/distilled water 1:1 3.3CYTODL\GNOSIS
Only those parts of the smears containing clusters of hepatocytes in monolayers and without too much admixture of blood should be examined. In a PAP smear, hepatocytes are well preserved and appear as polygonal cells with distinct cell borders. Their cytoplasm is interspersed with many small, red granules and larger, cloudy basophilic particles. The nuclei are regular in size and structiu-e and contain one or more distinct nucleoli and a fine, regular chromatine pattern. Different size classes (diploid, tetraploid and larger nuclei) can be recognized. Many binuclear cells are present (Fig. 4). Apart from many blood cells, the smears frequently contain cholangioductular cells appearing as branched cords of small cells with tightly packed cuboid or elongated nuclei smaller than hepatocyte nuclei. These bile ductules can be readily identified when the smears are stained for y-glutamyl transpeptidase (p. 25). In contrast to normal hepatocytes, they show a marked enzyme activity. Kupffer cells of various size are identified by their ingested material. Sometimes a sheet of mesothelial cells from the peritoneum can be seen. In PAP smears, much attention must be given to the interpretation of nuclear size and chromatin pattern. It is important, therefore, not to over- or under-stain the nuclei. This precaution is essential since nuclear hyper- or hypo-chromasia are important indicators for the diagnosis of prenecrotic or neoplastic conditions. Rat Uver cytology has been described in more detail by Boelsterli and Zbinden.^®" For comparison, human liver FNAB cytology is likewise well documented.""•^^· 26. 31-34. 54. 55. 59
Examples. (1) Male ZUR:SIV-Z rats (100 g initial weight) were given the hepatocarcinogen, N-nitrosomorpholine (NNM) (10 mg/100 ml drinking water) for 116 days. Dysplastic hepatocytes and hepatocellular carcinoma cells (Fig. 5) were
Fine-needle Aspiration Biopsy of Rat Liver 23 identified in FNAB smears and correlated with histopathological studies in the same animals.^^ (2) Male ZUR:SIV'Z rats (250 g) were treated with the mitogen, butylated hydroxytoluene (BHT) (500 mg/kg by gastric intubation for 9 days). Liver hyper trophy and hyperplasia was readily recognized (Fig. 6). (3) Male ZUR:SIV-Z rats (250 g) were injected the necrogenic hepatotoxin, carbon tetrachloride (1 ml/kg i.p. in oil, 48 h prior to FNAB sampling). Necrotic hepatocytes (pycnotic nuclei, fatty infiltration) were a common finding in the smears (Fig. 7). 4. Cytochemistry Cytochemical reactions performed on cryostat or paraffin-embedded histological sections can be adapted for FNAB smears. Care must be taken with regard to the type and duration of fixation and the specificity of the reaction (negative and positive controls).^ 4.1 TRIGLYCERIDE STAINING FOR DEMONSTRATION OF HEPATIC STEATOSIS
Accumulation of triglycerides in the liver occurs frequently as a consequence of toxic, metaboUc or nutritional disturbances and is, therefore, a common and important finding in chronic toxicity studies. Lipid accumulation is readily recognized in smears after staining with Fettrot 7B (Fig. 8). It can be scored semiquantitatively.^® " ^ ^Fixa smear for 5 min in 4Vo formaline (CaCOybuffered), containing 1 % CaCl — rinse with distilled water — ethanol35Vo (dip) — ethanol 70% (dip) — Fettrot 7B solution (I), 15 min uptolh — ethanol 70% (dip) — distilled water — Harris hematoxylin (II), 2 min — tap water, 10 min (several changes) — distilled water — mount in glycerol jelly. Triglycerides stain red. (I) Fettrot 7B stock solution: 0.5 g Fettrot 7B (Chroma, Stuttgart, FRG) dissolved in 100 ml isopropanol. Working solution: stock solution (6parts) + distilled water (4 parts), stand for 24 h, then filter (II) as described for Papanicolaou staining (p. 21). Example. Hepatic steatosis was induced in male ZUR:SIV-Z rats (200 g) by feeding a high fat diet for 2-16 days.^^ Mild to severe triglyceride accumulation was readily identified and showed good correlation with histological evaluation of the same liver lobes. 4.2 GLYCOGEN STAINING FOR DEMONSTRATION OF HEPATIC GLYCOGENOSIS
Apart from hereditary or alimentary disturbance of glycogen metabolism, glycogen storage is a feature concomitant with the development of neoplastic nodules in rat
24
υ . BOELSTERLI AND G. ZBINDEN
liver during experimental hepatocarcinogenesis.^'^ It must be emphasized, however, that minor alterations cannot be recognized due to physiological fluctuations of glycogen content as a function of circadian rhythm, nutritional state, topographic localization within the hepatic lobule, etc. Glycogen can be stained cytochemically by the periodic acid-Schiff (PAS) method (Fig. 9). — Fixa smear in ethanol, 5 min up to 24 h — dip slide into a 2% solution of celloidin in ether/ethanol 1:1 — dry for a few sec in air — 70% ethanol, 2 min — periodate solution (I), 10 min -- tap water, 10 min (several changes) — distilled water — Schiff reagents (II), 20 min — NaHSO^solution (III), 3x1 min — tap water (20 min), several changes — Harris hematoxylin (IV), 2 min — tap water, 10 min (several changes) — distilled water — 35% ethanol — 70% ethanol — ethanol abs, — ether/ethanol 1:1 (celloidin coat removed) — xylene, 1 min — xylene, 2 min — mount in Eukitt. Glycogen stains red. (I) periodate solution: dissolve 0.8 g periodic acid and 0.25 g sodium acetate in 20 ml distilled water, add 80 ml ethanol abs. (II) Schiff reagents (Merck, Darmstadt, FRG) (III) NaHSO^ solution: 5 ml NaHSO^ (10%) + 5mlHCl(lM) + 100 ml distilled water. Always prepare a fresh solution (IV) Harris hematoxylin as described for Papanicolaou staining (p. 21) 4.3 IRON STAINING FOR DEMONSTRATION OF HEPATIC SIDEROSIS
Iron loading of hepatocytes and/or Kupffer cells is demonstrated by iron staining of FNAB smears.^^' ^· This has recently become important as a negative marker for preneoplastic and neoplastic hepatocytes in rat hepatocarcinogenesis. These altered cells do not accumulate iron after long pretreatment with an iron-rich diet." — — — — —
Use iron-free glassware (rinse with HCl) fix smears in 4% neutral formalin/96% ethanol (1:9) for 1 min rinse in distilled water let dry in air stain in potassium ferrocyanide (10% solution in distilled water) for 5 min (prepare freshly) — add 0.5 parts of 10% HCl, stain for 30 min — rinse in distilled water — let dry in air
FIGS. 4-11. For captions see overieat.
FIGS. 4-11. Cytological smears of rat liver obtained by FNAB. (see overleaf) FIG. 4. Normal hepatocytes (p. 22 ). PAP staining. Regular polygonal cells with distinct cell borders and typical cytoplasmic staining with red granulation and cloudy basophilic particles. Regular nuclei with one or more distinct nucleoh. Note many binuclear hepatocytes. χ 400.
FIG. 5. Hepatocytes of a rat treated with iV-nitrosomorpholine (10 mg/100 ml in drinking water) for 116 days (p. 22 ). Apart from neoplastic nodules, hepatocellular carcinoma has developed. PAP staining. Irregular, basophilic cells featuring marked anisokaryosis and nuclear crowding. The hypochromatic nuclei harbour one or more distinct nucleoli of various size and shape, χ 400.
FIG. 6. Hepatocytes of a rat treated with butylated hydroxytoluene (500 mg(kg p.o. for 9 days) (p. 23 ). PAP staining. The hypertrophic hepatocytes are swollen and show huge polyploid nuclei with the typical chromatin pattern. The red cytoplasmic granulation which is present in normal hepatocytes is missing, χ 400.
FIG. 7. Hepatocytes of a rat treated with carbon tetrachloride (1 ml/kg i.p. in oil, 48 h prior to FNAB) (p. 23). PAP staining. A group of necrotic hepatocytes featuring cytoplasmic vacuolization, nuclear pycnosis, nuclear marginal chromatin condensation and hyperchromasia. χ 400.
FIG. 8. Liver cells of a rat fed a high-fat diet (p. 23) for 2 weeks. Severe hepatic steatosis has developed. Triglyceride droplets in hepatocytes are stained intensely red with Fettrot 7B X 310.
FIG. 9. Hepatocytes of a rat treated with 7V-nitrosomorpholine (10 mg/100 ml drinking water) for 84 days. Altered hepatocytes within neoplastic nodules are characterized by marked glycogen storage which is stained purple with the PAS reaction (p. 24 ). Note intranuclear glycogen, χ 400.
FIG. 10. Liver cells of a rat treated with N-nitrosomorphohne (10 mg/100 ml drinking water) for 28 days stained for y-glutamyl transpeptidase (7-GT) activity (p. 25). Apart from the cholangioductular cells (branched cords) which exhibit a marked 7-GT activity, the carcinogen-altered hepatocytes of preneoplastic foci or within the neoplastic nodules show a positive γ-GT reaction outlining the cell membranes, χ 250.
FIG. 11. Hepatocytes of a rat treated with 7V-nitrosomorpholine (10 mg/100 ml drinking water) for 70 days stained for glucose-6-phosphatase (G-6-Pase) activity (p. 26). In contrast to normal hepatocytes which show a diffuse cytochemical reaction in the cytoplasm but not in the nucleus, these carcinogen-altered cells display a focal intranuclear enzyme activity and in some cells only a weak or missing cytoplasmic G-6-Pase activity, χ 250.
Fine-needle Aspiration Biopsy of Rat Liver counterstain with 1% neutral red, 3 min rinse with distilled water and let dry mount in Eukitt. Iron stains blue.
25
4.4 GLUTAMYL TRANSPEPTIDASE STAINING FOR DEMONSTRATION OF PRENEOPLASTIC AND NEOPLASTIC HEPATOCYTES
Evidence is accumulating that a manyfold rise in 7-glutamyl transpeptidase (γ-GT) activity in rat hepatocytes during experimental hepatocarcinogenesis is an early and reUable marker for carcinogen-altered liver cells in preneoplastic foci, neoplastic nodules and hepatocellular carcinoma." ^* " 2® A positive enzyme reaction is observed in fetal and neonatal hepatocytes but not in normal adult rat liver.^ After exposure to a carcinogen hepatocytes featuring a positive enzyme reaction outhning the plasma membranes are readily identified (Fig. 10). Normal hepatocytes give a negative or only very faint color reaction. In all livers, cholangioductular cells display a marked y-GT activity (p. 22). The histochemical method of Albert et aU with y-glutamyl-a-naphthylamide as substrate is used. The liberated y-glutamyl moiety is accepted by glycylglycine, and the a-naphthylamine is coupled to Fast Garnet GBC, a diazonium salt, to form an insoluble red precipitate of diazo dye. Wash FNAB samples free from blood in ice-cold PBS (p. 20), transfer them onto a glass slide with a drop of PBS and prepare a smear. Fix immediately in ice-cold absolute ethanol for 10 min. Let dry in air. Slides can be stored at 4''C. — Encircle the fixed, air-dried cells on the glass slide with a fat pencil, place a few drops of substrate solution (III) on them and incubate for 30 min at 25 °C — rinse in 0.9% NaCl, 3x1 min — CuSO^(0.1M),2min — distilled water — Harris hematoxylin (IV), 1-2 min — tap water, 10 min (several changes) — distilled water — mount in glycerol jelly (I)
200 μmoles y-glutamyl-a-naphthylamide (= 5.45 mg) (Serva, Heidelberg, FRG) dissolved in 60 ml NaCl (0.9%) (II) 1 mmol glycylglycine (= 132 mg) dissolved in 40 ml phosphate buffer (0.1 M, pH6.7) (III) immediately before use, mix (I) and (II) and add 50 mg Fast Garnet GBC (Serva, Heidelberg, FRG), stir, filter (IV) Harris hematoxylin as described for Papanicolaou staining (p. 21). Example. Male ZUR.SIV-Z rats (170 g initial weight) were given the hepatocarcinogen, N-nitrosomorpholine (NNM) (10 mg/100 ml drinking water) for 56 days. Already after 1 week of treatment, about 50% of the animals were found to contain y-GT positive hepatocytes in FNAB smears, and after 4 weeks of treatment, almost all of the smears searched were positive." (See Fig. 10.)
26 υ . BOELSTERLI AND G. ZBINDEN 4.5 AMINO ACID NAPHTHYLAMIDASE STAINING FOR DEMONSTRATION OF BILE CANALICULI IN CHOLESTASIS
The cytochemical staining method for FNAB smears in human liver and that of rat and guinea pig is described in detail by Wasastjerna.^^ ^® 4.6 ALKALINE PHOSPHATASE STAINING
Increased alkaline phosphatase activity is, according to Ekelund,^^ a highly sensitive method for detecting mild bile stasis in smears. The cytochemical method for FNAB smears is described by Wasastjerna.^* 4.7 ADENOSINE TRIPHOSPHATASE (ATPase) AND GLUCOSE-6-PHOSPHATASE (G-6-Pase) STAINING
Focal loss of ATPase and G-6-Pase have been reported to occur in preneoplastic foci and neoplastic nodules of rat liver treated with various hepatocarcinogens.^^ *® The canalicular ATPase and the cytoplasmic G-6-Pase can easily be demonstrated by cytochemical methods (Fig. 11). However, as mentioned above, care must be taken in interpreting the results, as enzyme activity fluctuates in the rat Uver as to the site of the cells investigated within a hepatic lobule. The method of Wachstein and MeiseP® are appUed as modified by Rabes et al.*^ 4JA
ATPase — Wash FNAB samples in ice-cold sucrose/EDTA (0.25 M/O.OOl M), transfer them onto glass slides, prepare a smear, let dry — fix with glutaraldehyde (2.5Ψο in cacodylate buffer, p. 27) for 5 min. Rinse with buffer solution. Let dry. — encircle the fixed, air-dried cells on the glass slide with a fat pencil, place a few drops of substrate solution (I) on them and incubate for 15 min at37''C — rinse with distilled water — ammonium sulfide (1 % aqueous solution), 1 min — rinse with distilled water — mount in glycerol jelly. Site of ATPase activity stains brown-black (lead sulfide) (I) substrate-buffer solution: tris-maleate buffer (0.25 M, pH 7.2), MgSO^. 7H^O (5mM), Pb(NO^)2 (5mM), ATP.Na2(0.8 mM).
4.7.2 G-6-Pase Use the same procedure as for A TPase, except: — use unfixed smears — use glucose-6-phosphate.Na2 (0.8 mM) as substrate instead of A TP.Nai — after incubation with ammonium sulfide and rinsing with distilled water, postfix the smear in neutral 4Vo formalin for 5 min. (SeeFig. 11.)
Fine-needle Aspiration Biopsy of Rat Liver
27
5 Special Techniques 5.1 ELECTRON MICROSCOPY
Liver FNAB samples can be processed for electron microscopy by the usual technique.^2 Since the material often consists of very smaU fragments, rinsing and changing fixatives must be performed with great caution. The influence of the duration of glutaraldehyde fixation is evaluated by Hagelqvist" using FNAB samples of guinea pig hver and salivary gland. The penetration of the fixative into the tissue of needle biopsies from human Uver is studied by Petersen.*^ Expel the aspirated material in needle and syringe directly into a glutaraldehyde solution (ice-cold) (I), wash in cacodylate buffer (II), postfix in osmium tetroxide (III), dehydrate with dimethoxypropane^^ and embed in an epoxy resin as for routine electron microscopy. (I)
glutaraldehyde solution: 10 ml glutaraldehyde (25%), 50 ml cacodylate buffer (II), 30 ml bidistilled water, adjust to pH 7.3 and fill up with water to 100 ml (II) cacodylate buffer: 0.2 Af sodium cacodylate, pH 7.3 (III) 2% O5O4 in cacodylate buffer
5.2 FLUORESCENCE MICROSCOPY AND POLARIZATION MICROSCOPY FOR DEMONSTRATION OF DRUG-INDUCED PHOSPHOLIPIDOSIS
PhosphoUpidosis induced by amphiphilic drugs^' manifests itself as storage of myelin bodies in the lysosomes of hepatocytes. These can be recognized either in the polariza tion microscope, appearmg as *'Maltese cross" bodies (birefringent figures)" or, if the drug itself is a fluorochrome (as in the case of mepacrine) by fluorescence micro scopy."Wash FNAB samples free from blood in ice-cold PBS (p. 20), transfer them onto a glass slide with a drop of PBS and prepare a smear. Cover immediately with a coverslip and surround with nail varnish. Examine the slides in polarized light (oil immersion, 100 x objective) or in the fluorescence microscope (filter combina tion as for acridine orange fluorescence). Example. Male rats of the ZUR:SIV-Z strain (250 g) were treated with mepacrine (Geistlich, Wolhusen, Switzerland) (100, 200, 400 and 800 mg/kg respectively by gastric intubation for 2 days), or with chlorphentermine (CFS, Basel, Switzerland) (50 mg/kg by gastric intubation for 10 and 35 days respectively). A control group received water. The results were graded semiquantitatively and are Ipresented in Table II (0 no detectable fluorescent bodies/Maltese cross bodies; 1 + few and small bodies; 2 + small and large bodies in many cells; 3 + many bodies in all cells). The results indicate that the cellular inclusions were recognized in all smears and verified as myelin bodies (lysosomes) by electron microscopical examination (p. 27) of FNAB samples of the same animals. 5.3 ENZYMATIC DISSOCIATION AND CYTOFLUOROMETRY
Liver FNAB samples consist of smaU tissue cylinders of 1-2 mm up to maximaUy 10 mm in length. Apart from these grossly visible fragments, many small clusters of hepatocytes and even single cells are also aspirated and are especially abundant in
28
υ . BOELSTERLI AND G. ZBINDEN TABLE II. Semiquantitative evaluation of mepacrine- and chlorphentermine-induced myelin bodies in rat hepatocytes obtained by FNAB and investigated in cytological smears by fluorescence microscopy (FM) and polarization microscopy (PM) (Explanation of grading in the text, p. 27). control
FM PM
chloφhentermine (days treated with 50 mg/kg/d)
mepacrine dose (mg/kg/d for 2 days) 100
200
400
800
1+ 1+
1+ 1+
2+ 2+
3-f 3+
10
1+
35
1+
liver samples from rats treated with various hepatotoxic agents. Usually these fragments are smeared on glass slides for microscopical examinations. Cytofluorometric studies on smears, however, proved to be of minor value as the cells are not always present in monolayers and are intermingled with various blood cells. Thus, enzymatic dissociation of the FNAB specimens and further studies on cell suspensions should be preferred for cytofluorometric investigations of DNA,® enzyme activity (γ-GT)^^ or other assays. The effect of the negative pressure during aspiration biopsy on the viability of single cells was evaluated by Plesnicar et al.**^ 6 Biochemical Assays In addition to cytochemical studies (p. 23), enzyme activities can also be determined quantitatively in the liver FNAB homogenate. However, the activity cannot be ascribed to a certain cellular type, and the origin of the homogenized sample as to the location within the liver lobe remains obscure. Although the enzyme pattern is heterogeneous within the liver lobule (reviewed by Jungermann and Sasse^O good results have been obtained in FNAB studies of normal and pathological livers in man^^-^^ *® and in rodents.^ " In toxicity studies, enzymes involved in biotransformations (detoxification and activation) of xenobiotics are especially important and are described and discussed in detail later in this book. Of great value is the possibility of assaying several enzymes in the same homogenate. In order to express a specific enzyme activity, the measured activity should be related to the protein content of the homogenate. Literature 1. ALBERT, Z., J. ORLOWSKA. M. ORLOWSKI and A. SZEWCZUK (1964). Histochemical and biochemical investigations of gamma-glutamyl transpeptidase in the tissues of man and laboratory rodents. Acta Histochem. 18,78-89. 2. ALBERT, Z., Z. RZUCIDLO and H. STARZYK (1970). Comparative biochemical and histochemical studies on the activity of gamma-glutamyl transpeptidase in the organs of fetuses, newborns and adult rats. ActaHistochem, 37,34-39. 3. BANNASCH, P. (1968). The Cytoplasm of Hepatocytes during Carcinogenesis, Recent Results in Cancer Research, Vol. 19. Springer, Berlin, Heidelberg, New York. 4. BANNASCH, P. (1978). Cellular and subcellular pathology of liver carcinogenesis. Primary Liver Tumors (Edited by H. Remmer et al.), MTP Press, Lancaster, pp. 87-111. 5. BANNASCH, P. and H. ANGERER (1974). Glykogen und Glukose-6-Phosphatase wfthrend der Kanzerisierung der Rattenleber durch N-nitrosomorpholin. Arch, Geschwulstforsch, 43, 105-114. 6. BAUNSGAARD, P., G. C. SANCHEZ and C. J. LUNDBORG (1979). The variation of patiiological changes in the liver evaluated by double biopsies. Acta Path, Microbiol. Scand. A 87,51-57.
Fine-needle Aspiration Biopsy of Rat Liver
29
7. BERNE, C. and S. WESTMAN-NAESER (1978). Enzymatic studies of uver ceUs obtained by fineneedle biopsy from mice with the obese-hyperglycemic syndrome (genesymbol ob). Horm, Metab, Res. 10.124-127. 8. BICHEL, P., P. FREDERIKSEN, T. KJAER, P. THOMMESEN and L. L. VINDELÓV (1977). Flow microfluorometry and transrectal fine-needle biopsy in the classification of human prostatic carcinoma. Cancer Añ, 1206-1211. 9. BOELSTERLI, U. (1979). Gamma-glutamyl transpeptidase—an early marker for hepatocarcinogens in rats. Trends in Pharmacological Sciences 1, 47-49. 10. BOELSTERLI, U. and G. ZBINDEN (1978). The use of fine-needle aspiration biopsy for the diagnosis of hepatic steatosis in rats. Toxicology Letters 2,35-43. 11. BOELSTERLI, U. and G. ZBINDEN (1979). Application of fine-needle aspiration biopsy for the diagnosis of dysplastic and neoplastic liver cell changes induced by N-nitrosomorpholine in rats. Arch. Toxicol. Al, 225-238. 12. BOLL, I. (1969). Das Zytohepatogramm. Fortschr. Med. 87,420-422. 13. BRITS, C. J. (1974). Liver aspiration cytology. South Afr. Med. J. 48,2207-2214. 14. CAMERON, R., J. KELLEN, A. KOLIN, A. MALKIN and E. FARBER (1978). γ-Glutamyltransferase in putative premalignant liver cell populations during hepatocarcinogenesis. Cancer Res. 38, 823-829. 15. CARNEY, C. N. (1975). CUnical cytology of the liver. Acta Cytol. 19,244-250. 16. DANNMEIER, H. (1973). Die Feinnadelbiopsie der Leber. Verh. Dtsch. Ges. Path. 57, 243-246. 17. DOMINIS, M., S. CERLEK and D. SOLTER (1973). Cytology of diffuse Uver disorders. Acta Cytol. 17,205-208. 18. DULBECCO, R. and M. VOGT (1954). Plaque formation and isolation of pure lines with poliomyelitis viruses. / . Exp. Med. 99,167-182. 19. EKELUND, P. (1971). A cytological study of extra- and intrahepatic cholestasis in the rat. Ann. Med. Exp. Biol. Fenn. 49,49-53. 20. ELEQUIN, F. T., F. M. MUGGL\, N. A. GHOSSEIN and K. SCHREIBER (1977). A quick method for concentrating and processing cancer cells from serous fluids and fine-needle aspiration aspirates. Acta Cytol. 596-599. 21. G O S S N E R , W . and H. F R I E D R I C H - F R E K S A (1964). Histochemische Untersuchungen über die Glukose-6-Phosphatase in der Rattenleber wáhrend der Cancerisierung durch Nitrosamine. Z. Naturforsch. 19,862-863. 22. HAGELQVIST, E. (1978). Light and electron microscopic studies on material obtained by fine-needle biopsy. Acta Otolaryngol., Suppl. 354. 23. HANSEN, H. A. and A. WEINFELD (1959). Hemosiderin estimation and sideroblast counts in the differential diagnosis of iron deficiency and other anemias. Acta Med. Scand. 165,333-357. 24. HARADA, M., K. OKABE, K. SHIBATA, H. MASUDA, K. MIYATA and F. ENOMOTO (1976). Histochemical demonstration of increased activity of γ-glutamyl transpeptidase in rat liver during hepatocarcinogenesis. Acta Histochem. Cytochem. 9,168-179. 25. HENRIQUES, U. V. and K. HASSELSTRÓM (1977). Evaluation of jaundice: fine-needle aspiration hver cytology as a discriminating tool. Dan. Med. Bull. 24,104-108. 26. JOHANSEN, S. and J. MYREN (1971). Fine-needle aspiration biopsy smears m the diagnosis of liver diseases. Scand. J. Gastroenterol. 6,583-588. 27. JUNGERMAN, K. and D. SASSE (1978). Heterogeneity of Uver parenchymal cells. Trends in Biochemical Sciences 198-202. 28. KALENGAYI, M. M. R., G. RONCHI and V. J. DESMET (1975). Histochemistry of gamma-glutamyl transpeptidase in rat liver during aflatoxin B^-induced carcinogenesis. / . Natl. Cancer Inst. 55, 579-588. 29. LÜLLMANN, H., R. LÜLLMANN-RAUCH and O. WASSERMANN (1975). Drug-induced phospholiposes. Crit. Rev. Toxicol. 4,185-218. 30. LUNDIN, P., A. LUNDQUIST and O. LUNDVALL (1969). Evaluation of fine-needle aspiration biopsy smears in the diagnosis of liver iron overload. Acta Med. Scand. 186,369-373. 31. LUNDQUIST, A. (1970). Fine-needle aspiration biopsy for cytodiagnosis of malignant tumour in tiie liver. Acta Med. Scand. 188,465-470. 32. LUNDQUIST, A. (1970). Liver biopsy with a needle of 0.7 mm outer diameter. Acta Med. Scand. 188,471-474. 33. LUNDQUIST, A. (1971). Fine-needle aspiration biopsy of Üie Uver. Acta Med. Scand., Suppl. 520. 34. LUNDQUIST, A. and M. AÍKERMAN (1970). Fine-needle aspiration biopsy in acute hepatitis and liver cirrhosis. Ann. Clin. Res. 2,197-203. 35. LUNDQUIST, A. and P. OCKERMAN (1969). Fine-needle aspiration biopsy of tiie Uver in healtiiy adults. Activity of lysosomal acid hydrolases. Enzym. Biol. Clin. 10,300-304. 36. LUNDQUIST, A. and P. OCKERMAN (1970). Fine-needle aspiration biopsy of human Uver for enzymatic diagnosis of glycogen storage disease and gargoyUsm. Acta Med. Scand. 59, 293-2%.
30 υ . BOELSTERLI AND G. ZBINDEN 37. LUNDQUIST, Α., P. A. OCKERMAN and B. SCHERSTEN (1969). Fine-needle aspiration biopsy of the Uver in healthy adults. Activity of enzymes of glycogen utilization and glucose production. Enzym. BioL 10.8-12. 38. MENGHINI, G. (1970). One-second biopsy of the liver. New J, Med, 283, 582-585. 39. MULLER, L. L. and T. J. JACKS (1975). Rapid chemical dehydration of samples for electron micro scopic examination. / . Histochem Cytochem, 23,107-110. 40. OCKERMAN, P. A. (1%7). Glucose-6-phosphatase assay on microgram amounts of liver tissue. Clin, Chim, Acta 17,201-206. 41. OCKERMAN, P. A. (1968). A technique for the enzymatic diagnosis of glycogen storage disease on very small tissue specimens. Acta Paediat. Scand, 57,105-109. 42. PEARSE, A. G. E. (1968-1972). Histochemistry, Churchill Livingstone, Edinburgh, London. 43. PEDIO, G. (1976). Diagnose und Schnelldiagnose der Brustdrüsentumoren durch Feinnadelpunktion. Schweiz, Med, Wschr, 106,477-480. 44. PETERS, T. J. and C. A. SEYMOUR (1978). Analytical subcellular fractionation of needle-biopsy specimens from human liver. Biochem, J, 174,435-446. 45. PETERSEN, P. (1977). Glutaraldehyde fixation for electron microscopy of needle biopsies from human livers. Acta Path, Microbiol, Scand, A 85,373-383. 46. PLESNICAR, S., C. RUBIO, A. SIGURDSON and J. ZAJICEK (1968). Studies on the effects of aspiration biopsy on aspirated cells. Acta Cytol. 12,454-461. 47. RABES, H., P. SCHOLZE and B. JANTSCH (1972). Growth kinetics of diethyl-nitrosamine-induced enzyme-deficient ^'preneoplastic** liver cell populations in vivo and in vitro. Cancer Res, 32, 2577-2586. 48. SCHAUER, A. and E. KUNZE (1968). Enzymhistochemische und autoradiographische Untersuchungen wáhrend der Cancerisierung der Rattenleber mit Diáthyhiitrosamin. Ζ. Krebsforsch, 70, 252-266. 49. SCHAUER, A. and E. KUNZE (1976). Tumours of the Uver, Pathology of Tumours in Laboratory Animals, vol. 1, Tumours of the Rat, part 2 (Edited by V. S. TURUSOV), L\RC Sci. Publ. 6, Lyon, pp. 41-72. 50. SÓDERSTRÓM, Ν. (1966). Fine-needle aspiration biopsy, Ahnqvist & WikseU, Stockholm. 51. SOLOWAY, R. D., A. H. BAGGENSTOSS, L. J. SCHOENFIELD and W. H. J. SUMMERSKILL (1971). Observer error and sampling variability tested in evaluation of hepatitis and cirrhosis by liver biopsy. Am, J, Dig, Dis, 16,1082-1086. 52. SOOST, H. J. (1976) Lehrbuch derKlinischen Zytodiagnostik, Georg Thieme, Stuttgart. 53. STAIGER, G. R. (1974). Light-microscopic demonstration of drug-induced myelin bodies in the liver of the rat. Experientia 30,385-386. 54. STORMBY, N. and M. AKERMAN (1973). Aspiration cytology in the diagnosis of granulomatous Uver lesions. Acta Cytol, 17,200-204. 55. SZASZ, G., L. SAFRANY and A. LUX (1%9). Die Zytologie des Leberpunktats. Dtsch, Gesundheitswes, 22,1306-1311. 56. TATEMATSU, M., T. SHIRAI, H. TSUDA, Y. MIYATA, Y. SHINOHARA and N. ITO (1977). Rapid production of hyperplastic liver nodules in rats treated with carcinogenic chemicals: a new approach for an in vivo short-term screening test for hepatocarcinogens. GANN6$, 499-507. 57. VANDERLAAN, M., C. CUTTER and F. DOLBEARE (1979). How microfluorometric identification of Uver cells with elevated ganmia-glutamyltranspeptidase activity after carcmogen exposure. J. Histochem, Cytochem, 27,114-119. 58. WACHSTEIN, M. and E. MEISEL (1957). Histochemistry of hepatic phosphatase at a physiologic pH with special reference to the demonstration of bile canaliculi. Amer, J, Clin, Pathol, 27, 13-23. 59. WASASTJERNA, C. (1979). Liver, Aspiration Biopsy Cytology, part 2, Cytology of Infradiaphragmatic Organs (Edited by J. ZAJICEK) Karger, Basel, München, Paris, London, New York, Sydney. 60. WASASTJERNA, C. and P. EKELUND (1974). The amino add naphtiiylamidase reaction of the bile canaliculi in Uver smears. Acta Cytol, 18,23-29. 61. WASASTJERNA, C , P. REISSELL, J. KARJALAINEN and P. EKELUND (1972). Fatty Uver in diabetes. A cytological study. Acta Med, Scand, 191,225-228. 62. WILLL\MS, G. M. and R. S. YAMAMOTO (1972). Absence of stainable iron from preneoplastic and neoplastic lesions in rat Uver with 8-hydroxyquinoUne-induced siderosis. J, Natl, Cancer Inst, 49,685-692. 63. WILLIAMS, G. M., M. KLAIBER. S. E. PARKER and E. FARBER (1976). Nature of early appearing, carcinogen-induced Uver lesions resistant to iron accumulation. / . Natl, Cancer Inst, 57, 157-165. 64. WITTEKIND, D. and J. STAUBESAND (1970). Ueber farbstoffabhángige Variationen zeUularer Autophagie. I. Mitteilung: Neutrakot und Mepacrin. Cytobiologie 2,275-314. 65. ZAJICEK, J. (1974). Aspiration Biopsy Cytology, part I. Cytology of Supradiaphragmatic Organs. Karger, Basel. 66. ZAJICEK, J. (1977). Aspiration biopsy cytology. Lymphology 10,94-101.
Fine-needle Aspiration Biopsy of Rat Liver
31
67. ZAJICEK. J. (1979). Aspiration Biopsy Cytology, part 2. Cytology of Infradiaphragmatic Organs. Karger, Basel, München, Paris, London, New York, Sydney. 68. ZAJICEK, J., T. CASPERSSON, P. JAKOBSSON, J. KUDNYNOWSKI, J. LINSK and M. US-KRASOVEC (1970). Cytologic diagnosis of mammary tumors from aspiration biopsy smears. Comparison of cytologic and histologic findings in 2,111 lesions and diagnostic use of cytophotometry. Acta Cytol. 14, 370-376. 69. ZETTERBERG, A. and P. L. ESPOSTI (1976). Cytophotometric DNA-analysis of aspirated cells from prostatic carcinoma. Acta Cytol. 20,46-57.