s of papers DETERMINATION OF THE ANTITUMOR AGENT ADRIAMYCIN IN RAT TISSUE W.J. van Oort, B. Kerkhofs and A. van Dijk Adriamycin is an an thracycl ine der iva t ive c u r r e n t l y in widespread clinical use . It is a ve ry act ive d r u g in sev eral types of tumors as small cell lung cancer and breas t cancer . Cardiac toxic i ty is one of i ts most severe toxic side effects . T a r g e t t i n g adriamycin by encapsulat ion in liposomes, it is necessa ry to inves t iga t e in pharmacological exper i ments the d i s t r ibu t ion of adriamycin in plasma and s eve r al t ypes of t i s sue of r a t s . In addit ion to the determination of adriamycin in plasma (1), a t i s sue sample p re t reatment is p re sen ted , s t a r t i n g by f r eeze -d ry ing the t i s sues , degenera t ion of the cells by addit ion of AgNO.~, shak ing for 10 minutes , ad jus t ing the pH at 8.9 and e~t r a c t i n g by ch loroforml -heptanol (1+1, v / v ) . The foliowing s t eps are ident ical to the descr ibed HPLC method (1). Prel iminary d i s t r ibu t ion p a t t e r n s obtained by adminis t ration of free adriamycin and in two types of liposomes are p re sen ted . (1) A.M.B. Bots, W.J. van Oort , J. Noordhoek, A. van Dijk, S.W. Klein, Q.G.C.M. van Hoesel, J . ChromatoRT., accepted Pharmaceutical Labora tory , Cather i jnes ingel 60, Utrecht Academic Hospital, Univers i ty of Utrecht , Cathar i jnes ingel 101, Utrecht , The Nether lands THE BEHAVIOUR OF INJECTED SALTS IN REVERSED PHASE SYSTEMS -IMPLICATIONS FOR THE DETERMINATION OF THE HOLD-UP TIME O.A.G.J. van derHouwen, A. Hulsh~ff and A.W.M. Indemans The interaction of salts of alkalimetals with reversed phase columns was studied by chromatographic means. For the interpretation the chromatograms have been compared with those obtained on non-modified silicagel under the same conditions. The results indicate that on injection of these salts three peaks are obtained. These peaks are proven to be caused by the injected anions, the injected cations and buffer concentration pulse respectively. The retention of the anion peak and the buffer concentration pulse peak are explained by charge exclusion and well known concentration pulse propagation concepts. The implication of these phenomena for the use of salts for the ~etermination of the column void volume are discussed. Pharmaceutical Laboratory, Department of Analytical Pharmacy, State University of Utrecht, Catharijnesingel 60, 3511GH Utrecht, The Netherlands r98 Vol. 4 I982 Pharmaceutisch Weekblad Scientific Edition DETERMINATION OF THEOPHYLLINE IN SERUM AND ITS BINDING TO SERUM PROTEINS WITH ISOTACHOPHORESIS J.C.Reijenga, A.Gaykema, F.E.P.Mikkers ............................................... The concentration of free theophylline in serum was determined with isotachophoresis in the therapeutical range 0-20 mg/l. Serum was ultrafiltrated , the filtrate diluted and directly injected. The bidding of the drug to total pool serum proteins, albumin and orosomucoid was determined as 55, 44 and 12% respectivily. The effect of different parameters during filtration was investigated. Laboratory for Instrumental Analysis, Eindhoven University of Technology, P.O.Box 513 Eindhoven (The Netherlands) Differential Pulse I~larographlc 0uantitation of the Vasodilator Nitroprusside in Human Serum. O.R.Leeuwenkampf H.Jousma, E.J. van der Mark, A.Bult Initially the following procedure for serum analysis was employed: protein elimination with i.~4 perchloric acid (i : 4), treatment with active charcoal, centrifugation, membranefiltration and analysis of the resulting serum solution with high performance differential pulse pularography. However, the peaks were considerably lower and distorted in comparison with IM perchloric acid solutions of nitroprusside due to the formation of nitroprusside complexes with copper and other metals present in serum. After elimination of the proteins and the metals with 1.2M perchloric acid containing 1 mg/ml ferrocyanide, the peaks have the height expected in case of 100% recovery. A linear calibration curve was obtained for spiked serum. Detection limit: 15 ng/ml. Gorlaeus Laboratories, dept. of Pharmaceutical Analysis and Analytical Chemistry, subfaculty of Pharmacy, Leiden, The Netherlands. DATA HANDLING IN ISOTACHOPHORESIS J.C.Reijenga, W.van Iersel ............................................... The properties of the universal detector signal in isotachophoresis prohibit the use of standard chromatographic integration procedures. Therefor a computerized conversion is applied to auto~ati cally monitor the signal and give both qualitative and quantitative inforn~tion, so that full use is made of the linear dynamic range (I0000) of the separation method. The method is illustrated with the analysis of oxalate, glycolate, citrate and phosphate in urine. Several data reduction procedures can be used in this respect. Laboratory for Instrumental Analysis, Eindhoven Universf~:y of Technology, P.O.Box 513,Eindhoven (The Netherlands) Title: MULTI-CHANNEL U.V.DETECTION AS AN AID FOR PURITY CONTROL USING HPLC.

AIM: To discriminate between adenocarcinomas that are primary to the ovary and metastatic to the ovary, especially of colonic and breast origin, by immunohistochemistry, using stepwise discriminant analysis or a decision tree. METHODS: 312 routinely processed, formalin fixed tissue specimens were used. The tumours were divided into a learning set (n = 159), composed of primary tumours of ovary, breast, and colon, and a test set, comprising 134 metastases from these sites and an additional 19 primary ovarian carcinomas. The immunohistochemical panel was composed of antibodies against cytokeratin 7 (CK7) and 20 (CK20), CA125, vimentin, carcinoembryonic antigen (CEA), gross cystic disease fluid protein-15 (GCDFP-15), and the oestrogen receptor (ER). The staining results of the tumours were expressed as the product of the staining intensity and the percentage of positive tumour cells. Analyses were first performed on the learning set and then evaluated on the test set. RESULTS: Although the immunostaining patterns showed a considerable overlap between the three types of adenocarcinoma, the breast carcinomas were typically positive for GCDFP-15 and often for ER, and negative for vimentin. Ovarian carcinomas were always positive for CK7 and to a lesser extent for CA125. Colonic carcinomas showed prominent positivity for CEA and CK20, while no staining was seen for ER and vimentin. In discriminant analysis, six antibodies (alpha CK7, alpha CK20, alpha CA125, alpha CEA, alpha ER, and alpha GCDFP-15) appeared to be necessary for optimal classification: 89% of the learning set and 82% of the test set were classified correctly. In the decision tree, only four antibodies (alpha CK7, alpha CEA, alpha ER, and alpha GCDFP-15) were used to obtain a correct classification score of 89% for the learning set and 84% for the test set. CONCLUSIONS: Using a semiquantitative assessment of the immunostaining results by a restricted panel of six antibodies with stepwise discriminant analysis, 80-90% of the adenocarcinomas of colon, breast, and ovary can be correctly classified. Discriminant analysis is computer aided and therefore an easy method and for each case a probability value of the classification result is obtained. The intuitive decision tree method provides a slightly better result, requires only four antibodies, and offers a more practical method for the surgical pathologist.

To discriminate adenocarcinoma metastases originating from either colon or ovary, a panel of immunohistochemical markers was evaluated. For this purpose, paraffin sections from 157 primary and metastatic colonic and ovarian carcinomas were immunostained. These cases were divided into a learning group of 46 colonic and 54 ovarian carcinomas and a test group of 29 colonic and 28 ovarian carcinomas, including all metastatic tumors, among which were five with unknown primary site at the time of testing. The sections were immunostained with antibodies against carcinoembryonic antigen (CFA), cytokeratin 7 (CK7), cytokeratin 20 (CK20), CA 125, vimentin, and CA 19.9. Staining results were expressed as the product of staining intensity and percentage of positive tumor cells. Stepwise discriminant analysis was applied on the learning set to obtain a classification function for both tumors. The validity of the classification function was evaluated using the test set. There was considerable overlap in immunostaining for both tumor types, but colonic carcinomas were typically positive for CEA and CK20 and negative for CK7 and CA125. Ovarian carcinomas were typically positive for CK7 and CA125 and negative for CEA and CK20. In discriminant analysis, the best combination of markers appeared to be CK7 and CFA. Only one sample of the test group (2%) was misclassified. Taking learning and test groups together, 136 of the 157 samples (87%) were correctly classified with high posterior probability (PP > .8). However, from the 28 mucinous ovarian carcinomas, only 19 (68%) could correctly be classified with high PP. When excluding the nonmnucinous ovarian carcinomas from the analysis, overall 87 of 103 (84.5%) of the samples were correctly classified (PP > .8) with a combination of CEA, CK7, and also vimentin. From the 28 mucinous ovarian carcinomas, only two (7%) were misclassified, and four could not be classified with sufficient certainty. In neither analysis did CK20, CA125, or CA19.9 emerge as discriminatory parameters. Based on the same data, an intuitive flow chart was constructed with which 129 of 157 cases could be classified (only one falsely) without further statistical analysis. The five metastases with an at first unknown primary could, according to the follow-up, all be classified correctly with high PP. Most ovarian carcinomas, including the mucinous ones, can be discriminated with high probability from colonic carcinomas using a panel of three antibodies directed against CEA, cytokeratin 7, and vimentin.

Plate 5 In situ hybridization to human immunoglobulin light chain mRNA in paraffin-embedded lymphoid tissue. Plate 6 In situ hybridization to histone mRNA in lymphoid tissue. Plate 7 In situ hybridization to glyceraldehyde-3-phosphate dehydrogenase mRNA in formalin-fixed rat skeletal muscle. Plate 8 In situ hybridization detection of mitochondrial rRNAs in paraffin section of human kidney.

Abstract i>In situ hybridization is a powerful tool for the detection and localization of nucleic acid sequences in cells and tissue sections. In this way, the presence of specific DNA fragments as well as (messenger) RNA products can be demonstrated with preservation of morphology (1-3). As morphology may be impaired during in situ hybridization, additional information regarding the cells involved may be required, depending on the scientific or diagnostic problem being studied. Since a particular cellular phenotype is characterized by the presence of specific proteins or antigens, immunocytochemical staining is required to reveal it. This phenotype can be studied by careful comparison of adjacent tissue sections stained by in situ hybridization and immunocytochemistry respectively. However, a more accurate and easier method is to analyse both simultaneously in one single, double-stained section. Moreover, only this approach allows the study of individual cells.

Epstein‐Barr virus (EBV) has been demonstrated in the Reed–Stenberg cells and their mononuclear variants (Hodgkin cells; H‐RS cells) in a substantial number of Hodgkin's disease (HD) cases. Moreover, EBV can modulate both in vivo and in vitro the expression of several cellular genes, including lymphoid differentiation markers. Therefore we investigated, in 64 cases of HD, the relationship between the presence of EBV and the expression of lymphoid (CD45RB), T‐ (CD3, CD45RO), B‐ (CD20, MB2 antigen, CDw75), and myeloid‐cell lineage markers (CD15), and of activation markers (CD30, EMA, and the 115D8 antigen) on the H‐RS cells. EBV‐positive cases, as demonstrated by the presence of EBER‐1 and ‐2 RNA and LMP‐1 protein expression, showed a significant reduction in the expression on H‐RS cells of T‐cell lineage (CD3, p<0·02), B‐cell lineage (CD20, P<0·005), AND ACTIVATION MARKERS (ema; P<0·002 and the 115D8 antigen; P<0·001) as compared with EBV‐negative cases. No differences were found in the expression of CD15, CD30, CD45RO, CD45RB CDw75, or the MB2 antigen on H‐RS cells in EBV‐positive and EBV‐negative HD cases. Interestingly, in 11 cases of EBV‐negative HD, B‐as well as T‐cell lineage markers could be found on some H‐RS cells. These data suggest that EBV in H‐RS cells is able to down‐regulate the expression of T‐ (CD3) and B‐ (CD20) cell lineage markers and lymphoid activation markers (EMA and the 115D8 antigen). For this reason, the origin of H‐RS cells in HD, as studies by immunohistochemistry, cannot be discussed without taking into account the presence of EBV.

Forty-four cases of Hodgkin's disease (HD), mostly of the nodular sclerosing type, were investigated for the presence of Epstein-Barr virus (EBV) by polymerase chain reaction (PCR) and DNA and RNA in situ hybridization (DISH, RISH), as well as by immunohistochemistry for the detection of latent membrane protein-1 (LMP-1) of EBV. In situ hybridization (ISH) was combined with immunohistochemistry to correlate the presence and activity of the virus at the cellular level. In 18/34 (53 per cent) cases, EBV-DNA sequences could be detected with the PCR method. In 12/18 positive cases, DISH and RISH were also positive. In the remaining six EBV-PCR positive cases, two were also positive with RISH and LMP-1, whereas no positive signal with DISH could be obtained. All DISH and/or RISH positive cases were also positive for LMP-1. With RISH, not only the Reed-Sternberg cells and their mononuclear variants (RS cells) stained positive, but also small and intermediate cells frequently reacted with the EBV-specific probes (EBER-1 and -2). Double staining with cellular markers (CD3, CD20, CD45, CD45RO, CD68, and the lectin PNA) revealed that most of the smaller EBER-positive cells frequently did not express T, B, or histiocytic markers, but that they, as well as the RS cells, showed cytoplasmic and membranous staining with PNA. These smaller EBER-positive cells were not found in EBV-PCR negative HD. EBER-positive RS cells were almost always LMP-1 positive, as well as a substantial proportion of the intermediate-sized cells, whereas the majority of the small EBER-positive cells remained LMP-1 negative.(ABSTRACT TRUNCATED AT 250 WORDS)

AIMS:To evaluate the expression of c-myc and bcl-2 oncogene products in Reed-Sternberg cells in Hodgkin's disease, especially in relation to Epstein-Barr virus infection and expression of EBV encoded latent membrane protein (LMP).METHODS:Tissues from 33 cases of Hodgkin's disease were studied for the presence of EBV DNA by polymerase chain reaction (PCR) and DNA in situ hybridisation (DISH), for the presence of EBER-1 and EBER-2 EBV RNA by RNA in situ hybridisation (RISH); and for the presence of LMP, bcl-2, and c-myc proteins by immunohistochemical staining.RESULTS:A substantial number of Reed-Sternberg cells expressed bcl-2 in 20 of 29 (69%) and c-myc in 30 of 32 (94%) Hodgkin's disease samples. In 18 of the 25 (72%) cases Reed-Sternberg cells expressed both oncogene products. Of these 18 cases, 10 (56%) were EBV-PCR positive; eight (44%) were EBV-PCR negative.CONCLUSIONS:Reed-Sternberg cells in Hodgkin's disease frequently express both bcl-2 and c-myc oncogene products, suggesting that these oncogenes may act in concert in the pathogenesis of the disease. Moreover, the expression of c-myc and bcl-2 proteins in Reed-Sternberg cells is independent of EBV and LMP status.

Silver-intensification methods described in the literature for the diaminobenzidine (DAB) and diaminobenzidine-nickel (DAB/Ni) endproduct of the peroxidase reaction were compared in model systems after immunoperoxidase and in situ hybridization. First, these methods were compared in immunohistochemical model systems, using the demonstration of glial fibrillar acidic protein (GFAP) and prostate-specific antigen (PSA) in paraffin sections of human brain and prostate tissue, respectively. When DAB without Ni was used as substrate, tissue argyrophilia caused considerable background staining. Only when this tissue reactivity was quenched with, e.g., CuSO4 with H2O2 or thioglycolic acid, were the results acceptable. A considerable improvement in the signal-to-noise ratio could be obtained when nickel was included in the substrate mixture. The methods that proved to be best for demonstration of GFAP and PSA made use of acid developer solutions. Subsequently, these methods were compared with other sensitive immunostaining methods for demonstration of the gamma-delta T-cell receptor in frozen lymphoid tissue. In this model a considerable increase in the number of positive cells could be obtained using silver intensification. The different methods using DAB/Ni were also compared for use in DNA in situ hybridization (DISH). In this case two model systems were used: human papilloma virus type 11 (HPV-11) DNA in condyloma tissue (abundant target model) and Epstein-Barr virus (EBV) DNA in a mononucleosis lymph node (low target model). For demonstration of HPV-11, all methods gave more or less satisfactory results, which were best with the acid developer solutions. Moreover, for demonstration of EBV DNA, a signal could be obtained only with these developer solutions. Such a method also proved suitable in double immuno-hybrido stainings for the demonstration of EBV DNA in specific antigen-positive Reed-Sternberg cells in paraffin sections of Hodgkin lymph nodes.

We studied 44 cases of Hodgkin's disease for the presence of Epstein‐Barr virus (EBV) DNA, its localization and the expression of the EBV receptor on the tumour cells. EBV DNA was found in 52% (16/31) of the Hodgkin's lymphomas using the polymerase chain reaction. With a very sensitive non‐radioactive DNA in situ hybridization technique in combination with immunohistochemistry for CD 30 or CD 15 antigens, EBV DNA was localized to Reed‐Sternberg cells and its mononuclear variants. The relationship between the presence of EBV DNA and the expression of the EBV‐receptor CR2 (CD 21) on Reed‐Sternberg cells was studied using the same techniques and two different monoclonal anti‐CD 21 antibodies. CR2 could be detected on a substantial number of the Reed‐Sternberg cells in EBV DNA positive Hodgkin's lymphomas (9/12; 75%), whereas in EBV negative cases positivity with anti‐CD 21 was rare (1/13; 8%). The results indicate that CR2 expression on Reed‐Sternberg cells and the presence of EBV DNA sequences are frequently associated in Hodgkin's lymphomas.

Human papillomavirus (HPV) can be detected in, and is probably involved in the etiology of, the majority of anogenital neoplasias. Infection with the virus induces a number of events in the infected epithelial cells that may lead to the development of benign or malignant tumors. One change that can be detected in the infected cells is in squamous differentiation, which is reflected by the pattern of cytokeratin polypeptide expression. By studying this pattern in relation to the presence of the virus, an indication may be obtained of the influence of the virus on the cellular differentiation in individual cells. By using a combination of DNA in situ hybridization and immunohistochemistry, for HPV and cytokeratin polypeptides, respectively, we studied the presence of HPV6 or HPV11 in condylomata accuminata derived from anogenital skin in relation to the cytokeratin polypeptides K1, 4, 8, 10, 14, and 18. We found that in many samples the presence of the skin-type cytokeratins K1 and K10 was decreased, whereas K13, and to a lesser degree K4, appeared. The cellular localization of these aberrations in cytokeratin expression could be related to the presence of HPV6 or 11 DNA in the tissue.

In the bronchoalveolar lavage of sarcoidosis patients the mononuclear cell infiltrate was enumerated on T helper and suppressor lymphocytes as well as macrophages by means of a triple-staining assay on cytospin slides. As was seen on the slides, lymphocytes were often adhered very closely to macrophages. This phenomenon, many times described but not understood, was studied in a group of 13 sarcoidosis patients, of whom 7 received prednisolone treatment. It could be shown that treatment with the corticosteroid was followed by an increase in the percentage of suppressor lymphocytes adhered to macrophages. Second, the number of such alveolar T suppressor lymphocyte-macrophage aggregates was dramatically increased in the prednisolone-treated patients.

Twenty-nine human immunodeficiency virus (HIV)-infected patients with white, nonremovable lesions on the lateral border of the tongue, clinically suggestive of oral hairy leukoplakia (HL), were studied. In particular, the value of local antifungal therapy in establishing the diagnosis of HL was investigated. In 15 patients (52%) the lesions could be ultimately attributed to a candidal infection of the tongue. In 10 of the remaining 14 patients, a biopsy was obtained from lesions persisting after local antifungal treatment. In all biopsy specimens, the diagnosis of HL was confirmed by histopathologic examination and the demonstration of Epstein-Barr virus DNA by polymerase chain reaction, Southern blot hybridization, and DNA in situ hybridization. The present data confirm that the diagnosis of HL in HIV-infected patients cannot be reliably made on clinical criteria alone, but requires histopathologic confirmation including the demonstration of Epstein-Barr virus DNA, preferably by DNA in situ hybridization. However, with regard to the differential diagnosis of white, nonremovable lesions on the lateral border of the tongue in HIV-infected patients, the present study suggests that persistence of lesions after local antifungal therapy is highly suggestive of HL.

The presence of human papillomavirus (HPV) and Epstein-Barr virus (EBV) was analyzed in 21 oral biopsy specimens of HIV-infected patients using the polymerase chain reaction (PCR) method. Biopsies were categorized as hairy leukoplakia (HL) (n = 12), candidiasis (n = 3), oral warts (n = 2), and clinically normal epithelium (n = 4). For HPV detection a modified general primer-mediated PCR method (GP-PCR), which detects a broad spectrum of HPV genotypes at sub-picogram levels, was used. Human papillomavirus DNA was only found in two oral warts and was identified as HPV type 32. Epstein-Barr virus DNA was detected in 16 biopsy specimens, including the 12 HLs, 2 cases of candidiasis, and 2 samples of normal epithelium. Epstein-Barr virus positivity in HL could be confirmed by Southern blot analysis and DNA in situ hybridization using biotinylated DNA probes (bio-DISH). Epstein-Barr virus bio-DISH was also positive in one sample of normal epithelium from a patient with HL. The results indicate that HL is strongly associated with EBV and not with any of the common HPV types that react with general HPV primers in the PCR. However the detection of EBV in normal oral epithelium by PCR and bio-DISH suggests that the presence of this virus is not exclusively related to HL.