ANALYSIS OF LEUCOCYTES IMMUNOCYTOCHEMISTRY

BY COMBINED CYTOPLASMIC

D.M. Swirsky Department of Haematological

Medicine,

ENZYME CYTOCHEMISTRY

AND MEMBRANE

Cambridge

Smears of washed leucocytes from a variety of acute myeloid leukaemias were stained for myeloperoxidase (4-chloro-naphthol) and with a variety of esterase substrates (naphthol AS-D chloroacetate, alpha-naphthyl butyrate, alpha-naphthyl acetate and naphthol AS-D acetate) followed by inhibition of endogenous peroxidases and then a two step immunoperoxidase process using a monoclonal anti HLA-DR antibody. This technique permits the simultaneous visualisation of HLA-DR positivity and enzyme staining in individual cells. HLA-DR positive myeloperoxidase positive cells are commonly found in acute myeloblastic leukaemia, but HLA-DR positive chloroacetate cells only rarely. Co-existence of Auer rods and HLA-DR positivity has been found. In acute myelomonocytic and monoblastic leukaemias the relationship between enzyme staining and HLA-DR expression is less clear. Details of the method and its limitations will be discussed. Conventional classifications of acute myeloid leukaemia based on morphology and cytochemistry have recently been augmented by the introduction of monoclonal antibodies directed against cell lineage specific antigens, e.g. glycophorin, platelet glycoproteins. The technique described in this presentation may contribute to further understanding of lineage commitment and differentiation in leukaemic states, and conversely help in the characterisation of new monoclonal antibodies that react with sub-populations of leucocytes.

IMMUNOHISTOCHEMICAL EVIDENCE FOR MULTITARGET DISTRIBUTION AND INTERACTION OF NEURAL ENKEPHALIN, NEUROTENSIN, SUBSTANCE P AND VASOACTIVE INTESTINAL POLYPEPTIDE IN THE MAMMALIAN HEART E. Weihe, M. Reinecke and W.G. Forssmann Department of Anatomy III, University of Heidelberg,

W. Germany

There is increasing immunohistochemical, radioimmunological and chromatographic evidence for the occurrence of cardiovascular active neuronal peptides in the heart. The aim of this study was a comparison of the distribution of cardiac nerves containing immunoreactivities for enkephalin (ENK), Substance P (SP), Neurotensin (NT) and vasoactive intestinal polypeptide (VIP). Key-anatomical areas of hearts from guinea pig, rat, cat, dog and tupaia were analysed with the light microscope using the peroxidase-antiperoxidase technique on consecutive sections of Bouin-fixed tissue. Immunoreactivities for ENK, NT, SP, VIP occurred in distinct nerve fibres and different distribution patterns. Partial co-distribution but not co-location of the immunoreactive (IR) peptides in identical nerve fibres was observed. NT-IR nerves supplied all segments of the coronary vasculature, atrial and ventricular myocardiocytes, sinuatrial and atrioventricular nodal cells. VIP-IR fibres occurred predominantly in the atria and abundantly in sinuatrial nodes where they formed numerous contacts with nodal cells. They were more prevalent in the coronary microvasculature and, like NT-IR fibres, formed frequent contacts with endothelial cells of capillaries and of pericytic venules. SP-IR fibres were predominantly found supplying larger coronary vessels. SP-IR fibres were frequently observed in interstitial, epicardial, endocardial and valvular locations whereas NT-IR and VIP-IR nervous supply to these areas of the heart was sparse. Cardiac ENK-IR nerve fibres were mostly restricted to intracardiac ganglia where they surrounded ganglionic cell bodies.. Cardiac ganglia contained VIP-IR cell bodies and a few VIP-IR fibres, abundant NT-IR and less commonly SP-IR fibres. NT, SP and VIP-IR fibres occurred also in cardiac chemoreceptors. The relation of the characteristically distributed peptidergic cardiac nerves to each other and to multiple targets indicates complex functional interactions in the extrinsic and intrinsic neuronal control of cardiac performance. 1983 Chapman and Hall Ltd. 7

A HIGH SENSITIVITY CHEMISTRY

POST-EMBEDDING

TECHNIQUE FOR ELECTRON MICROSCOPE

G.R. Newman, B. Jasani and E.D. Williams Dept. of Pathology, Welsh National School of Medicine,

IMMUNOCYTO-

Cardiff

The recently formulated acrylic resin LR White is an embedding agent for biological tissue which is very suitable for electron-immunocytochemistry. When polymerised, the plastic is hydrophilic, thus differing from epoxides and polyesters. Unlike methacrylates, ultrathin sections of LR White are quite beam-stable, and can be mounted unsupported on nickel grids. High resolution electron microscopy can be achieved with routinely fixed tissue embedded in LR White. However for electron immunocytochemistry, requiring good preservation of antigenicity, we have avoided the use of post-fixation in osmium. We have found a buffered mixture of highly purified monomeric glutaraldehyde and picric acid (BGPA) to be very effective in preserving both general ultrastructure and antigenieity. The resin will also accept tissue directly embedded from 70% alcohol, with improvement in immunostaining. By the direct application of a sensitive and versatile hapten sandwich immunoperoxidase procedure to ultrathin sections on nickel grids, excellent results, using antibodies to both polypeptide hormones and immunoglobulins, have been achieved. Diaminobenzidine used in conjunction with hydrogen peroxide as the substrate in peroxidase techniques has a low electron-density. Currently, this is intensified by the use of osmium tetroxide; however, in both pre- and post-embedding immunotechniques, tissue osmiophilia leads to unavoidable increases in general background density. Because of this, and of other problems inherent in the use of osmium, we have explored the use of a range of other metal compounds, of which the most effective was gold chloride, for the intensification of DAB. We will present results of a semi-quantitative study using antibodies to two polypeptide hormones.

A QUANTITATIVE PROCEDURES

COMPARISON OF THE SENSITIVITY OF THREE IMMUNOPEROXIDASE

B. Jasani, D.A. Millar and E.D. Williams Dept. of Pathology, Welsh National School of Medicine,

SANDWICH

Cardiff

The sensitivity of an immunocytochemical procedure has been defined as,"the lowest amount or concentration of tissue antigen which, with some assurance, can be distinguished from background". The somewhat loose wording of this definition has been necessary firstly because there is as yet no single independent means of determining accurately the antigen content of tissue sites being localised, and secondly because so far the majority of workers in the field have resorted to purely subjective means for grading the intensity of tissue staining. An artificial step-wedge section system, recently described by us, provides a useful means of overcoming the above difficulties. In essence, the system allows the highly reproducible incorporation of varying concentrations of a known antigen into steps of gelatin. All of these can be conveniently congealed into a single block which, when frozen, yields high quality serial sections. Immunoperoxidase staining of these sections is not only consistently uniform and reproducible, but is very amenable to quantitative microdensitometric analysis. Using a human IgG step-wedge, we have compared the sensitivity of biotin-avidin, PAP and DNP-hapten immunoperoxidase sandwich procedures. The minimum antigen concentrations detected by the three techniques were 2.5, 0.15, and 0.08 mg/ml gelatin respectively. The lowest value was calculated to represent as little as 77.6 femtograms of antigen per unit volume of the section subjected to microdensitometrie measurement. We therefore suggest that the sensitivity of an immunocytochemical technique should be re-defined on a quantitative basis.

IMMUNOCYTOCHEMICAL LOCALISATION OF THE N-TERMINAL FLANKING PEPTIDE OF THE HUMAN CALCITONIN PRECURSOR IN NORMAL C CELLS AND IN MEDULLARY CARCINOMA OF THE THYROID 1 2 1 A. Ali-Rachedi, I.M. Varndell, C.J. Hillyard , R.K. Craig , I. Maclntyre and J.M. Polak 1 2 Dept. of Histochemistry and Endocrine Unit, RPMS, London; Middlesex Hospital, London The structure of the human calcitonin precursor mRNA has recently been revealed after cloning of the eDNA. In human pro-calcitonin mRNA, the sequence coding for calcitonin is flanked on both sides by sequences coding for additional peptides. The predicted C-terminal flanking peptide has been shown to circulate in plasma and to display calcium-lowering activity and has thus been named Katacalcin. We report here the immunocytochemical localisation of the N-terminal flanking peptide by region-specific antibodies recognising a mid-portion sequence (PYE-14). The materials consisted of medullary carcinoma of the thyroid (n = 4) and normal human thyroid (adult: n = 2; foetal: n = 2). In view of the extensive sequence homologies between PYE and its rat counterpart, rat thyroid was also used (n = 4). Tissues were fixed in para-benzoquinone or formaldehyde vapour or in formalin solution. The peroxidase-antiperoxidase (PAP) method was used. Antisera to PYE were obtained from rabbits immunised with synthetic PYE coupled to keyhole limpet haemocyanin. Parallel immunostaining for calcitonin was performed to ascertain the presence of C cells. Appropriate controls were performed. Numerous PYE-immunoreactive cells were found in all medullary carcinomas and foetal thyroids, fewer in adult thyroid. Rat thyroid was also found to contain numerous PYE-immunoreactive cells, although the intensity of the immunostaining was weaker. Absorption experiments showed that all immunostaining could be prevented by the prior addition of I nmol/ml of PYE while large concentrations (up to 20 nmol/ml) of calcitonin, Katacalcin, somatostatin and ACTH had no effect. It is concluded that the PYE-immunoreactivity found in both normal and tumour C cells is due to the presence of the N-terminal flanking peptide of pro-calcitonin as predicted from the sequence of the mRNA.

AN IMMUNOPEROXIDASE CYTOLOGICAL SMEARS

TECHNIQUE FOR THE DETECTION OF GENITAL HERPES INFECTION ON

R. Adams and D. Springall Regional Cell Path. Serv., Wandle Valley Hospital,

Mitcham Junction,

Surrey

Genital herpes is currently "spreading like wildfire ~' both throughout the female population and the media. Present methods of detection of the disease include tissue culture and virus isolation methods as well as cases picked up during the routine screening of cervical smears for malignancy. The sensitivity of the latter method is questionable - borne out by studies comparing virus isolation and cytopathological results. Both tissue culture and virus isolation method, though sensitive, are consuming in terms of time and resources. An immunoperoxidase method is described here for the routine detection of cells infected by the herpes virus (HSV2). The method is rapid (approx. 3.5 hours), inexpensive, not requiring complex apparatus~ moreover comparing favourably in terms of sensitivity with the methods currently available. Optimum fixation (type of fixative and time) and antibody dilution combine to give a reliable technique for use on cytological smears prepared in a similar way to those for routine gynaecological investigation. The time saved in the direct staining of smears rather than cultured cells is advantageous to clinician and patient alike - positive result being rapidly obtained.

OF CERVICAL INTRAEPITHELIAL NEOPLASIA O.A.N. Husain, K.C. Watts, J. Gauntlett, A. To, M. Silverstone, R. Fray and J. Taylor-Papadimitriou 1 Dept. of Cytopathology, Charing Cross Hosp.;l ICRF, Lincoln's Inn Fields, London The present histological and cytological interpretation of cervical abnormalities is subjective and discrepancies often arise in the diagnosis of particular lesions. A technique is required which would resolve these problems by providing an objective distinction between the degree of C.I.N. and invasive carcinoma. Furthermore it could provide an additional parameter which may be of value in the automation of cervical cancer screening. Immunocytochemistry has been used in cellular pathology to assist in the diagnosis and detection of neoplasia (i). Monoclonal antibodies have been raised against a delipidated preparation of Human Milk Fat Globule and designated HMFG-I and HMFG-2. These antibodies were used for defining the stages of differentiation in normal and malignant cells in breast lesions. A preliminary study of the histological distribution of these markers by the immunoperoxidase technique in normal and neoplastic tissues has indicated that these antibodies have a potential for the characterisation of normal and malignant epithelial cell types (2). We have evaluated this potential on histological and cytological preparations of the cervix using the indirect immunoalkaline phosphatase technique. Cytological preparations from 123 patients and histological sections from 75 patients have been investigated. The staining pattern was compared to the histological or cytological diagnosis and the results have revealed a quantitative association between the expression of HMFG-I and/or HMFG-2 and the changing pattern from squamous metaplasia onward through the C.I.N. lesions of increasing severity. We found in fact that staining occurs in both the endocervical cells and in the metaplastic squamous cells of the transformation zone but not in the native squamous cells of the ectocervix. These findings indicate that HMFG-I and HMFG-2 may be of value in elucidating the processes involved in the carcinogenesis of cervical intraepithelial neoplasia but may have some limitations in the realm of automation. (i) To et al (1982) Am. J. Clin. Path. 78:214-219 (2) Arklie et al (1981) Int. J. Cancer 28:23-29

INCREASES IN STRIATAL D 2 DOPAMINE RECEPTORS AND DECREASES IN CHOLECYSTOKININ AND SOMATOSTATIN IN TEMPORAL LOBE IN RELATION TO THE TYPES I AND II SYNDROME IN SCHIZOPHRENIA T.J. Crow, T.E. Adrian, S.R. Bloom, A.J. Cross, I.N. Ferrier, E.C. Johnstone, Y.C. Lee, D. O'Shaughnessy, F. Owen, D.G.C. Owens, J.M. Polak and G.W. Roberts C.R.C., Northwick Park Hosp., Harrow, and R.P.M.S., Hammersmith Hosp., London It has been suggested (Crow, T.J.,1980, B.M.J., 280: 66-68) that two syndromes can be identified in schizophrenia on the basis of the distinction between positive and negative symptoms - a reversible neurochemical disturbance responding to neuroleptic drugs (the type I syndrome) and an irreversible component related to structural changes in the brain (the type II syndrome). The neurochemical correlates of the two syndromes have been investigated in post-mortem brain in patients who had been assessed in life for positive and negative symptoms with the following findings: (i) D 2 dopamine receptors are increased, and the increase is related to the presence of positive symptoms; (ii) cholecystokinin immunoreactivity is reduced in hippocampus and amygdala and somatostatin immunoreactivity in the hippocampus of patients with negative symptoms; (iii) VIP (Vasoaetive Intestinal Polypeptide) is increased in the amygdala in relation to positive symptoms.

I0

DISTRIBUTION

OF PEPTIDES

IN THE HUMAN AMYGDALA

G.W. Roberts I, T.J. Crow, S.R. Bloom I and J.M. Polak I Div. of Psychiatry, C.R.C., Northwick Park Hosp., and IR.P.M.S.,

London

Using radioimmunoassay, large quantities of peptides have been reported in the amygdala. However, exact localisations of the peptides within this region are unknown. We have studied the distribution of seven peptides (VIP, CCK-8, substance P, neurotensin, methionine enkephalin, neuropeptide Y (NPY) and somatostatin) within this region using immunocytochemistry. Five brains were obtained 3-5 hours post-mortem and fixed in 0.4% para-benzoquinone in phosphate buffer. After dissection, 20-micron cryostat sections were cut and used for immunocytochemistry (PAP method). Large numbers of immunoreactive fibres were found in the central (VIP, CCK-8, substance P, neurotensin), medial (CCK-8, substance P) and lateral (VIP) amygdaloid nuclei. NPY and somatostatin-positive fibres were found scattered in all regions. NPY and VIP-positive cell bodies were located in the lateral nucleus, whilst CCK-8 and substance P cell bodies were found in the medial and central nuclei. VIP, NPY, CCK-8 and somatostatin-containing neurones and scattered substance P and neurotensin fibres were present in the temporal cortex adjacent to the amygdala. Each peptide was present in fibres within the stria terminalis and in the ventral amygdalofugal pathway. The discrete localisation of peptides and their presence in afferent/efferent pathways implies functional importance. Peptides may play a direct role in the transmission/modulation of information passing between amygdala and hypothalamus.

PRESENCE OF NEUROPEPTIDE-CONTAINING

FIBRES IN THE HUMAN CORPUS CALLOSUM

G.W. Roberts 1, T.J. Crow, S.R. Bloom I and J.M. Polak 1 Div. of Psychiatry, C.R.C., Northwick Park Hosp., and IR.P.M.S.,

London

The corpus callosum is a large commissural pathway composed of fibres originating in cortical regions and projecting across the midline to terminate in a homotopic fashion in the contralateral cortex. Recent radioimmunoassay studies have reported the presence of CCK-8 in the corpus callosum and surgical transection of the corpus callosum in animals implies the existence of CCK-8 fibres which reach across the midline and terminate homotopically in the contralateral cortex. Because of the hypothesised importance of the corpus callosum in higher cognitive functions in humans, we investigated the human corpus callosum to determine if peptide-containing fibres were present within it. Five brains were obtained 3-5 hours post-mortem and fixed in 0.4% para-benzoquinone in phosphate buffer. After dissection, 20-micron cryostat sections were cut and used for immunocytochemistry (PAP method). CCK-8, N-PY, VIP and somatostatin-containing fibres were found to be present within the corpus callosum. No substance P, methionine enkephalin or neurotensin immunoreactive fibres were detected. CCK-8 and VlP-containing fibres were found in moderate numbers and these fibres could be traced entering the corpus callosum from dorsal cortical regions (particularly cingulate gyrus), crossing the midline and exiting into the contra-lateral cortex. Somatostatin and NPY-containing fibres were found in greater numbers and were present in all portions of the corpus callosum. NPY and somatostatin-positive neuronal cell bodies in the deep cortical layers (V and VI) were observed to give rise to fibres which entered the corpus callosum. Large numbers of NPY and somatostatin positive neuronal cell bodies were found clustered around the corona radiata and close to the lateral surface of the corpus callosum, fibres from which were observed to cross the midline and exit into the contra-lateral cortex. Our findings of peptides in the corpus callosum may imply functionally significant actions in higher cognitive processing and an important role in inter-hemispheric communication between homotopic cortical regions. ii

EFFECTS OF THYROID STATUS ON NEUROPEPTIDE

DEVELOPMENT IN RAT BRAIN

1 P.L. Woodhams , G. McGregor, J.M. Polak and S.R. Bloom i M.R.C. Developmental London

D. O'Shaughnessy,

M. Blank, T.E. Adrian, Y.C. Lee,

Neurobiology Unit, London,

and R.P.M.S.,

Hammersmith Hosp.

The effects of neonatal thyroid deficiency or hyperthyroidism on the development of neuropeptide systems in the rat brain was examined at two weeks of age. The levels of vasoactive intestinal polypeptide (VIP), cholecystokinin octapeptide (CCK), substance P (SP), somatostatin and neurotensin were measured in five brain areas by radioimmunoassay, and immunostained cell bodies were evaluated quantitatively. Concentrating particularly on the cingulate gyrus of the cerebral cortex and the peptides found there in intrinsic neurones, in hypothyroidism both the concentration of VlP and the number of VIP-positive cells were significantly decreased. In contrast to these effects on the relatively late-maturing VIP neurones, the earlier developing somatostatin system was relatively unaffected, whilst peptides such as SP and neurotensin, which are found in cortical fibres rather than cells, were found by radioimmunoassay to be elevated. Hyperthyroidism had less marked effects than neonatal thyroidectomy, although the concentration of CCK but not the number of immunostained cell bodies was elevated. The results indicate that thyroid disorders produce disynchronous shifts in the developmental patterns of the different neuropeptides and that the effects of thyroid hormone on peptides as on other transmitter systems are critically dependent on the developmental profile of the system in question.

THE DISTRIBUTION

OF PLACENTAL ALKALINE PHOSPHATASES

A.A. Epenetos ICRF Labs., Lincoln's

Inn Fields,

IN GERM CELL TUMOURS

London

Two monoclonal antibodies to placental alkaline phosphatase (HI7E2 and D20L) were used in an indirect immunoperoxidase reaction on fresh frozen tissues to detect the phenotypic heterogeneity of germ cell tumours, other tumours and normal tissues. All germ cell tumours gave a strongly positive reaction with HI7E2 (these included seven seminomas, three trophoblast malignant teratomas, two intermediate malignant teratomas, two undifferentiated malignant teratomas and two mixed teratoma and seminoma). On the other hand, only 10% of the cells of three seminomas reacted positively with antibody D20L. With regard to non germ cell neoplasms two out of three ovarian carcinomas and one uterine carcinoma produced a positive reaction with HI7E2. Also three out of five colon carcinomas produced a weak and patchy reaction with both antibodies. All normal tissues (including testis, cervix, ovary, lung) except placenta, produced a negative reaction with both antibodies. It is concluded that antibody HI7E2 can be used as an aid in the differential diagnosis of testicular germ cell tumours provided that the patchy and weak reaction with some colon carcinomas is borne in mind. It is interesting that antibody D20L also against placental alkaline phosphatase showed a narrower distribution found only on some seminomas. This finding may be accepted by the complex nature of placental alkaline phosphatases and may help in the understanding of the pathogenesis of germ cell tumours.

12

DEMONSTRATION OF DONOR SPECIFIC ANTIBODIES BINDING TO RENAL ALLOGRAFTS REJECTION USING AN INDIRECT IMMUNOPEROXIDASE TECHNIQUE

IN VASCULAR 1

L.P.Trickett~

P.R. Evans, A.G. Maclver,

Wessex Regional Immunology Unit, Portsmouth

Service,

C. Spencer, J.L. Smith and M. Slapak

Southampton,

and

1 Wessex Regional Transplant

The role of donor specific antibodies in the vascular rejection of renal allografts has been studied by investigating the ability of serum samples from 15 graft recipients to stain frozen sections of preanastomosis biopsies of donor kidneys by the indirect immunoperoxidase (IP) method. Seven out of 15 recipients have shown positive staining with varying patterns. Peritubular and glomerular capillary endothelial staining was observed in sera from five recipients and in the acid eluates from three failed grafts. Sera and graft eluates from two recipients stained tubular cells, one showed proximal tubule and brush border staining. Histological and IP examination of post-transplant biopsies from all seven recipients were consistent with vascular rejection. Four of the eight recipients whose sera did not stain their donor kidney have currently surviving grafts with episodes of predominantly cellular rejection; vascular rejection was established as the cause of rejection in only one of the four grafts that failed. Data will be presented on the development and specificity of antibodies binding to donor kidney and their correlation with both clinical outcome and routine IP examination of biopsies subsequent to transplantation.

THE LOCALISATION

OF RENIN IN THE ARTERIES OF THE HUMAN KIDNEY

1 G.B.M. Lindop

1 , T.T. Downie

i , J.A. Stewart

2 , P. Corvol

2 , J. Menard

3 and A.F. Lever

1 3 2Dept. of Pathology and M.R.C. Blood Pressure Unit, Western Infirmary, Inserm U36, 75005, Paris, France

Glasgow;

Renin is synthesised and stored in large amounts in the granular cells of the juxtaglomerular apparatus from where it enters the blood. Blood-borne angiotensin II (its active product) probably contributes to the control of blood pressure. However, smaller amounts of renin are more widely distributed in other arteries, and, since the enzyme which converts angiotensin I to angiotensin II has also been found in arteries, angiotensin II may be formed and act locally within the artery wall without entering the circulation. Using the peroxidase-antiperoxidase technique with an antibody to pure human renin, we found that the cells in the human juxtaglomerular apparatus which stain most strongly are situated on the outer aspect of the arterioles. We found that the renin-containing cells in the larger arteries in the kidney are also externally situated, mainly in the outer ring of the media. With ultrastructural immunocytochemistry we have shown that these cells contain renin storage~granules and we have also immunostained renin in the lumen and on the membranes of the rough endoplasmic reticulum. This we present as evidence that these renin-containing cells in renal arteries distant from the juxtaglomerular apparatus also synthesise and store renin. The external situation of these renin-containing cells is clearly functionally important.

13

THE IMMUNOCYTOCHEMICAL

DEMONSTRATION

OF RENIN IN A JUXTAGLOMERULAR

CELL TUMOUR

G.B.M. Lindop, T.T. Downie and J.A. Stewart Dept. of Pathology, Western Infirmary, Glasgow Juxtaglomerular cell tumours are rare renal tumours which present with hypertension due to the secretion of renin. Using an antibody to pure human renin we have studied the distribution of immunoreactive renin in a juxtaglomerular cell tumour. We used the peroxidase-antiperoxidase technique on paraffin and resin-embedded sections and showed that most of the immunostainable renin was contained in the plump granular spindle cells around the blood vessels in the tumour. The fine structure of juxtaglomerular tumour cell granules is identical to that of the granules of normal juxtaglomerular cells. There are various morphological types: rhomboidal crystalline structures, round membrane-bound granules and forms which are intermediate in shape. Using ultrastructural immunocytochemistry we demonstrated immunoreactive renin both in the crystalline and in the round membrane-bound granules as well as in intermediate forms. Occasional cells contained some granules which did not stain for renin. Their nature is uncertain. In this study we have established for the first time the renin content of these various types of granules in the human renin-secreting cell.

IMMUNOHISTOCHEMICAL

LOCALISATION OF DESMIN IN SURGICAL PATHOLOGY i

G. Coggi, P. Dell'Orto, IV Dept. of Pathology, Center, Siena, Italy

i P. Leoncini,

P. Neri and G. Viale

Univ. of Milan Medical School, Milan,

Italy,

and

I Research

The intermediate filament protein desmin (MW 53kd) has been considered as a marker for smooth and striated muscle cells. Desmin purified from uterine leiomyomas has been used to raise a polyclonal antiserum in guinea pigs. This antiserum was employed to localise desmin in formalin-fixed and paraffin-embedded human tissues by the avidin-biotinylated peroxidase complex method. Desmin immunostaining was achieved both in smooth and striated normal muscle cells. In the former, the reaction product was diffusely distributed throughout the cytoplasm, whereas in the latter the cross-striations were sharply immunostained. Similarly, desmin-like immunoreactivity was observed in muscle cells in a large number of cases of different hyperplastie and neoplastic conditions (lymphangiomyomatosis of the lung, leiomyomas, leiomyosarcomas and rhabdomyosarcomas). Desmin appears to be a useful cytochemical marker for recognising the muscular origin of benign and malignant tumours. This holds particularly true with regard to polymorph rhabdomyosarcomas, whose conventional histological diagnosis rely upon the time-consuming and often unreliable search for cytoplasmic cross-striation in the neoplastic cells. The immunohistochemical localisatien of desmin allows a fast and precise identification of rhabdomyoblastic cells and therefore it is a valuable tool in the differential diagnosis of highly undifferentiated large cell tumours.

14

COMPARISON OF DIRECT METHODS OF IMMUNOCYTOCHEMISTRY AND ELISA USING AVIDIN-BIOTIN COMPLEX (ABC) TECHNIQUE TO INDIRECT IMMUNOCYTOCHEMISTRY AND RADIOIMMUNOASSAY WITH MONOCLONAL ANTIBODIES (MoAb) BINDING HUMAN LUNG CANCERS J. Mulshine, F. Cuttitta, M. Bibro, M. Matthews, S.-M. Hsu, A. Gazdar and J. Minna NCI-Navy Medical Oncology Branch, COP/DCT/NCI/NIH and Naval Medical Center, Bethesda, MD 20814, USA Three well characterised murine monoclonal antibodies generated against human lung cancers were biotinylated and analysed in three assay systems to assess the value of direct binding assays detected by ABC reagent for work with MoAb. Ten established human lung cancer cell lines [five small cell lung cancers (SCLC) and five non-small cell lung cancer (NSCLC)] were used in the assays shown below. Murine Monoclonal Antibodies 534F8 (antiSCLC) I03D4 (antiNSCLC) 704AI (antiNSCLC) SCLC/NSCLC SCLC/NSCLC SCLC/NSCLC RIA [MoAb (ug/ml)] # lines positive

0.i 4/2

0.3 0/4

0.7 0/4

Direct ELISA [MoAb (ug/ml)] # lines positive

0.7 4/2

1.8 0/4

1.8 0/4

Indirect ICC [MoAb (ug/ml)] # lines positive

5 4/3

I0 0/4

i0 0/3

Direct ICC ~MoAb (ug/ml)] # lines positive

i0 4/3

25 0/4

25 0/2

From these experiments we conclude the results of direct assays correlated closely with those of indirect assays. Using biotinylated primary MoAb in direct immunostaining and ELISA with the ABC reagent sensitivity was diminished at a specific protein concentration. This could be compensated for by increasing the concentration of the MoAb without compromising specificity. Since direct assays require less time to perform and preserve accuracy these techniques have broad potential applications.

QUANTITATION IN IMMUNOCYTOCHEMISTRY E. Heyderman, G. Powell and T.C. Richardson Dept. of Histopathology, St. Thomas's Hospital Medical School, London Where immunohistological techniques are used to demonstrate intracellular antigens such as immunoglobulins, comparison of different methods of fixation, reagents and staining schedules may be made by counting the number of positive cells. This can be carried out using a microscope graticule or more sophisticated image analysis apparatus. In most publications, where a comparison of intensity of staining is made, results are subjectively scored, for example + to +++. We are investigating the use of a Reichert-Univar micro-spectrophotometer in combination with a HewlettPackard HP-85 computer to assess differences in staining intensity and distribution of the reaction product. Preliminary data will be presented on a comparison between two different sheep anti-rabbit indirect peroxidase conjugates prepared in this laboratory, and commercially available avidin-biotin peroxidase complex (Vectastain). 15

1 B.L. Mepham and P.G. Isaaeson Dept. of Exptl. Pathology, Southampton Gen. Hosp., Morbid Anatomy, University College, London

1

Southampton,

and

Dept. of

Alpha-l-antitrypsin is now recognised as an immunohistochemical marker of histiocytes (macrophages, monocytes) and malignant tumours derived thereof. It has been shown to be synthesised by these cells and is seen as abundant cytoplasmic granules, sometimes characteristically condensed in the Golgi region adjacent to the nudleus or in a perinuclear ring. Demonstration of alpha-l-antitrypsin after fixation in neutral buffered formalin (NBF) is reliable providing that antigenic reactivity has been unmasked using proteolytic enzymes (trypsin). However fixation in other fixatives particularly those containing mercuric chloride has produced unreliable staining results in that specific staining intensity was greatly reduced or indeed absent. Blocks of normal human lung were fixed in several mercuric chloride containing fixatives and also NBF. A model system was also prepared using pellets of cells made from normal human monocytes or cultured malignant histiocytes (U937) and were fixed in a similar manner. All blocks were then processed to paraffin wax. Three micron sections were stained by the immunoperoxidase-PAP technique but those that had been fixed in NBF were treated with trypsin prior to immunostaining. In formalin fixed trypsin-treated sections alpha-l-antitrypsin was demonstrated in lung macrophages, blood monocytes and malignant histiocytes. Whereas in sections from the mercury fixed tissues alpha-l-antitrypsin was not apparent due to an overall brown colouration of all tissue structures. When mercury fixed sections were treated with trypsin prior to immunostaining the intensity of the overall brown staining was reduced but alpha-l-antitrypsin was not demonstrated.

IMMUNOHISTOCHEMICAL PLASIA

STUDY OF FOLLICLE CENTRE CELL LYMPHOMA AND FOLLICULAR HYPER-

H.F. AI Saffar Southampton General Hospital,

Southampton

Using conventional antisera and monoclonal antibodies, IP techniques were applied to frozen sections of reactive and neoplastic lymphoid tissues. The following features were found to assist in the differential diagnosis between follicular hyperplasia and follicular lymphoma: I. Surface Ig phenotype: Monotypia of the lymphoma cells was a useful marker confirming the diagnosis of FCC lymphoma. 2. Surface Ig staining patterns: The reactive follicles were characterised by two distinct features: i. a well-defined follicle mantle; ii. intercellular dendritic staining with antisera to immunoglobulins and to complement. In most cases of follicular Cb/cc lymphoma these two features were absent, although a number of different staining patterns were observed. 3. T-cell content and distribution: Both follicular hyperplasia and follicular Cb/cc lymphoma showed a high content of T-cells. T-cells in most reactive germinal centres were more abundant in the upper zone, while in Cb/cc lymphoma they were scattered uniformly throughout the neoplastic follicles. In diffuse FCC lymphomas, centrocytic lymphoma always contained a very small percentage of T-cells, whereas diffuse Cb/cc and Cb lymphoma showed a higher proportion of T-cells. 4. ~ recePt0rs: Ell monoelonal antibody was used to demonstrate C 3 receptors of follicular dendritic reticulum cells. This antibody showed a high specificity for lymphoid follicles whether reactive or neoplastic. Ill-defined groups of dendritic reticulum cells were detected by Eli staining in most diffuse follicle centre cell lymphoma.

16

HISTOCHEMICAL AND IMMUNOHISTOCHEMICAL HUMAN STOMACH

OBSERVATIONS

1

F. Malchiodi

ON INTESTINAL METAPLASIA OF THE

1

, F. Carlei, P. Mingazzini

1

1

, A. Covotta

2

, E. Lezoche

and J.M. Polak

2

II Istituto di anatomia e istologia patologica and VI e clinica chirurgica, Universita di Roma, Italy; Dept. of Histochemistry, R.P.M.S., London Intestinal metaplasia is a common feature of chronic gastritis. The most characteristic feature is the presence of mucin-containing goblet cells of intestinal type. An additional important feature is the presence of non-secretory columnar cells bearing a prominent striated border of microvilli. On the basis of the presence of absorptive cells in place of mucin-secreting cells amongst goblet cells it is possible to distinguish complete and incomplete intestinal metaplasia. Depending upon the presence of sulphated or non-sulphated mucins, colonic and small intestinal types of intestinal metaplasia have been classified. The purpose of our study was to investigate the effect of intestinal metaplasia on endocrine cell population in the antrum and an attempt was made to correlate the type of metaplasia to the endocrine cell types present. Endoscopic biopsies from 22 patients with chronic gastritis and intestinal metaplasia have been studied histologically, histochemically and immunohistochemically. It was observed that different types of metaplasia were always present in single specimens. Absorptive and mucin secreting cells, and sulphated and non-sulphated mucins were found to be randomly distributed throughout the same gland or in different fields of the same specimen. On the basis of these results we could not make any distinction between different types of metaplasia. Besides gastrin and somatostatin cells normally present in antral mucosa, we could detect GIP-, CCK-, enteroglucagon- and secretin-containing cells. We conclude that intestinal metaplasia affects the endocrine cell population as well as epithelial cells since intestinal types of peptide-producing cells were identified.

NEURON-SPECIFIC ENOLASE IMMUNOREACTIVITY CARCINOMA OF THE LUNG

IN CYTOLOGICAL MATERIAL

FROM SMALL CELL

D.R. Springall, M.N. Sheppard, S. Van Noorden and J.M. Polak Dept. of Histochemistry, R.P.M.S., Hammersmith Hospital, London Neuron-specific enolase (NSE) is an isoenzyme of enolase. It has been found to be a good immunocytochemically detected marker of neuroendocrine cells and nerves throughout the body and tumours derived from them, including lung carcinoid and the more common and malignant small cell carcinoma of the lung. Small cell lung carcinoma is sensitive to radiotherapy and chemotherapy rather than surgery and accurate cytological diagnosis is therefore important, but is often very difficult. We have therefore attempted to use NSE immunostaining to detect small cell carcinoma cells in cytological smears and sections of wax-embedded cell buttons prepared from serous fluids as well as endoscopic biopsies, confirming the diagnosis by electron microscopy. Five fixatives (formol saline, Bouin's fluid, glutaraldehyde, methanol, acetone) were employed on smears, both wet-fixed and air-dried, which were then dried or stored in buffered saline. Cell buttons were fixed in formol saline or methanol and embedded in wax. Antibody to rat brain NSE was raised in rabbits and characterised by absorption with antigen. The best results for NSE immunostaining were obtained with smears wet-fixed in methanol or formol saline for 20 min. and stored wet. Air drying before fixation destroyed all immunoreactivity. Sections of cell buttons had much weaker NSE immunoreactivity than smears from the same case. Cells from all other lung tumour types examined were negative. We therefore suggest that NSE immunostaining may be a valuable marker for lung small cell carcinoma in cytological preparations, and further that such preparations could be even better than tissue setions. 17

DIRECT BIOTINYLATION OF ANTI-BOMBESIN MONOCLONAL ANTIBODY 2AII (MoAb 2All): WITH AVIDIN-BIOTIN COMPLEX (ABC) FOR ELISA AND IMMUNOHISTOCHEMICAL ANALYSIS

USE

F. Cuttitta, J. Mulshine, J. Fedorko, T. Moody, D. Papermaster and J.D. Minna NCI-Navy Medical Oncology Branch, COP/DCT/NCI/NIH and Naval Medical Center, Bethesda, MD 20814, USA -I0 MoAb 2All is a mouse IgG immunoglobulin which has a high affinity (Kd = i0 M) for the C-terminal end of bombesin (amino acids 8-14). The antibody has previously been shown to react with bombesin and related peptides (alytensin, litorin, ranatensin and GRP) but failed to react with substance P, eledoisin, physalaemin, neurotensin, leucine enkephalin, calcitonin, VIP, and somatostatin. MoAb 2All was directly biotinylated by a modification of the Heitzmann and Richards technique (Heitzmann & Richards, 1974, PNAS, 71: 3537-3541) using biotin-N-hydroxysuccinimide ester (BNHSE). The biotinylation reaction was carried out as follows: 1.2 mg of BNHSE was reacted with i mg MoAb 2All in phosphate-buffered saline (PBS) for 30-60 minutes at room temperature. Unreacted BNHSE was removed by pressure dialysis or neutralised by the addition of 10 mg BSA. The biotinylated antibody was assessed for solid phase peptide binding using ABC-peroxidase and was shown to retain its bombesin specificity. When comparing titration end-points on solid phased bombesin, the direct system (biotinylated primary antibody) was eight times less sensitive than using indirect detection (biotinylated secondary antibody), however gave similar results to PAP. We have used this biotinylated reagent at the light and electron microscope level for the immunohistochemical localisation of bombenergic cells expressed in human small cell carcinoma of the lung.

THE USE OF RETROGRADE TRACING TECHNIQUES IN COMBINATION WITH IMMUNOHISTOCHEMISTRY IN STUDIES OF VISCERAL AFFERENT PATHWAYS K.A. Sharkey, R.G. Williams and G.J. Dockray The Physiological Laboratory, University of Liverpool, Liverpool The existence of extensive systems of peripheral peptidergic nerves is now well established, but the precise projections are not as well understood. We have used a combination of immunohistochemistry and retrograde tracing with the fluorescent dye True Blue to study the afferent innervation of visceral structures, namely urinary bladder, stomach and parotid gland, with particular emphasis on substance P immunoreactivity (SP-LI). Five days after injection of the True Blue into these tissues fluorescent cell bodies were identified in sensory ganglia in rats fixed by perfusion with paraformaldehyde. Some fixatives, e.g. parabenzoquinone, which are good for preservation of immunoreactive peptides are not however, suitable for fixation of True Blue. Following injection of True Blue into the stomach, labelled cell bodies were observed in spinal ganglia at levels T7-L2, a high proportion (about 50%) of which contained SP-LI. Injection into the wall of the bladder revealed fluorescent cell bodies in spinal ganglia at levels TI2-L2 and L6-SI, but only 5-15% of these cells contained SP-LI. Unilateral injection into the parotid revealed labelled cells in the ipsilateral trigeminal ganglion, 5-10% of which contain SP-LI. Conclusions: I. Retrograde tracing combined with immunohistochemistry has revealed the afferent SP-LI innervation of visceral structures. 2. The proportion of afferent neurons containing SP-LI varies between tissues.

18

TEMPORAL CHANGES IN PANCREATIC ISLET A, D AND PP CELL POPULATIONS MENT OF OBESITY IN THE ZUCKER FATTY (fa/fa) RAT D. Gapp, l.J. Turkenkopf and M.R.C. Greenwood Depts. of Biology, Hamilton College, Clinton and Vassar College, USA

DURING DEVELOP-

Poughkeepsie,

NY,

Pronounced changes in topographical and numerical relationships among pancreatic islet populations are characteristic of a number of obesity and diabetes condtions. Studies with murine models of obesity and diabetes indicate that the extent of perturbation in islet cell relationships is directly related to the severity of the syndrome. The Zucker fatty (fa/fa) rat is a normoglycaemic, hyperinsulinaemic and hyperlipidaemic model of obesity. Perturbations in fatty rat islet cell populations have been reported, but these changes have not been quantified or analysed temporally. Pancreases from 20 day, 35 day and 5 month old lean (+/?) and obese (fa/fa) animals were divided into regions, fixed in Bouin's and embedded in paraffin. Serial 5 micron sections were stained for insulin, glucagon, somatostatin and pancreatic polypeptide by the PAP method. One slide from each region from each animal was selected for morphometric analysis. Islets from 20 and 35 day lean and obese and 5 month lean animals exhibited a normal mantle layer of non-B cells. Islets from 5 month fa/fa animals showed a greatly reduced mantle layer and exhibited a significant (two-fold) increase in mean islet area which was attributable to increased B cell mass. Volume densities of A, D and PP cells were unchanged in 20 and 35 day fa/fa islets except for a 30% increase in A cell volume density in 20 day fa/fa islets which coincided with an observed increase in pancreatic and plasma insulin content and thus may be significant. At five months A, D and PP cell volume density in fa/fa islets had dropped to one half or less of the values in the litter mate controls. Zucker fatty rat islets show little alteration in islet cell populations other than the increased B cell mass common to most rodent obesity/diabetes models. Contrary to ob/ob and db/db mouse islets, there appeared to be no increase in non-E cell mass in response to the expanding and hyperactive B cell mass. The mild changes in the islet cells of the fa/fa rat seem to reflect its normoglycaemic status and is on the other end of the spectrum from the severely hyperglycaemic, severely affected C57BL/Ks ob/ob or db/db mouse models.

SOME IMMUNOCYTOCHEMICAL APPLICATIONS ADRENALINE IN TUMOUR PATHOLOGY

OF ANTIBODIES TO SEROTONIN,

NORADRENALINE,

AND

1

A.A.J. Verhofstad and H.W.M. Steinbusch i Dept. of Anatomy and Embryology, Univ. of Nijmegen and Dept. of Pharmacology, University of Amsterdam, Netherlands

Free

Epithelial cells containing serotonin, noradrenaline or adrenaline can now be demonstrated by immunocytochemical techniques employing antibodies raised by immunogens consisting of the respective haptens linked to bovine serum albumin (i). In the present report it will be demonstrated that these antibodies might be useful tools in demonstrating serotonin-, noradrenaline- or adrenaline-immunoreactive cells also in tumours. The original procedure was based upon the use of cryostat sections of tissues taken from experimental animals, fixed by perfusion or immersion with 4% paraformaldehyde in 0.1M sodium phosphate buffer, pH 7.3. However, tumours submitted for analysis supposedly derived from serotonin-, noradrenaline- or adrenaline-containing cells have often not been fixed properly or have been embedded in paraffin. It will be shown that serotonin-immunoreactivity is preserved quite well in routinely fixed and paraffin-embedded tumour samples, allowing retrospective studies on previously collected material. (1) Verhofstad et al. (1983), pp. 143-168 Immunocytochemistry: (Wright, Bristol). 19

G.W. Roberts 12, T.J. Crow I, S.R. Bloom 2 and J.M. Polak 2 iDivision of Psychiatry, London

C.R.C., Northwick Park Hospital,

and 2Hammersmith Hospital

Appreciable quantities of neuropeptides have been found in the human hippocampus by radioimmunoassay, however little is known of their exact distribution in the hippocampus or their relationship with anatomical structures. We report here on an immunocytochemical study of the distribution of seven peptides, VIP, CCK-8, substance P, neurotensin, methionine enkephalin, neuropeptide Y (NPu and somatostatin within the hippocampus. Tissue was obtained 4-6 hours post-mortem and fixed in 0.4% para-benzoquinone. Cryostat sections were cut and processed for immunocytochemistry using the PAP method. Numerous VIP, CCK-8, NPY and somatostatin positive neuronal cell bodies were stained. VIP and CCK-8 containing cell bodies were predominantly found in the stratum moleculare and the stratum radiatum. NPY and somatostatin-positive cell bodies were found mainly in the stratum oriens and dentate gyrus. Neurotensinpositive cell bodies were found in the subiculum. Each of the peptides was found in nerve fibres within the hippocampus. Whilst VIP, CCK-8, NPY and somatostatin fibres were widely distributed, neurotensin, substance P and methionine enkephalin fibres showed a restricted distribution, being found close to the pyramidal layer of the CA1 and CA2 regions. Fibres containing each of the peptides were found running within the fornix and fimbria. CCK-8 and VIP have potent excitatory actions on hippocampal neurones, although the exact function of these and other peptides in the hippocampus is unknown. The fact that the peptides show a differential distribution within the hippocampus suggests that peptides play an important part in the organisation of hippocampal activity.

NEUROFILAMENTS

IN MERKEL CELL TUMOURS

G. Viale, J. Gu I and J.M. Polak 1 IV e Department of Pathology, University of Milan Medical School, Milan, iDepartment of Histochemistry, R.P.M.S , Hammersmith Hospital, London

Italy, and

Cutaneous Merkel cell tumours are uncommon neoplasms first described in 1972 as trabecular cell carcinomas. Owing to the ultrastructural identification of electron-dense neurosecretory granules in the cytoplasm of the neoplastic cells, these tumours have been recognised as neuroendocrine carcinomas probably originating from cutaneous Merkel cells. Moreover, recent studies have shown NeuronSpecific Enolase (NSE)-immunoreactivity in the neoplastic cells, thus confirming their neuroendocrine nature (Gu et al., 1983, Cancer, in press). Immunocytochemical investigations on intermediate filaments would represent another valuable tool at the light microscope level for the differential diagnosis of Merkel cell tumours and for the recognition of their histogenesis. Using the PAP-complex method we found the finest cytoplasmic immunoreactivity for 200K neurofilaments in paraffinembedded Merkel cell tumours. The immunoreactivity was confined to the cytoplasm of the neoplastic cells and it was particularly strong in the paranuclear area, the location and shape of which correlated well with the ultrastructural identification of a paranuclear clustering of criss-crossed intermediate filaments displacing the cytoplasmic organelles and neurosecretory granules. The immunolocalisation of neurofilaments appears to be a reliable method for diagnosis purposes. Moreover it provides further evidence for the neural origin of these peculiar tumours.

20