Flow cytometric detection of EBV (EBER snRNA) using peptide nucleic acid probes.

The application of peptide nucleic acid (PNA) probes for detection of Epstein-Barr Virus (EBV) snRNA in fixed cells is described. Fluorescein labelled PNA probes were used to detect EBER1 and EBER2 snRNA in Raji, Daudi and HS-Sultan cells. The fixation and permeabilization of cells were optimized. The optimal fixation was found to be 5% acetic acid plus 4% paraformaldehyde in PBS and the optimal permeabilization 0.5% Tween 20 in PBS whereas no proteolytic digestion was needed. The hybridization time needed with the PNA probes was only 1 h. When running mixed samples of Ramos (EBV neg.) Raji, Daudi and HS-Sultan (EBV pos.) cells in flow cytometry a strong fluorescence signal was seen in Raji, Daudi and HS-Sultan cells whereas no fluorescence signal was seen in the Ramos cells. In total 0.5% EBER positive Raji cells could easily be identified in a mixture of Raji and Ramos cells. The results were verified by fluorescence microscopy. It is concluded that PNA probes can be used for in situ hybridization in solution and the analysis can be done using flow cytometry or fluorescence microscopy. PNA probes therefore may facilitate and enhance the potential use of the in situ hybridization/flow cytometry combination.

Reactivities of 20 anti-human monoclonal antibodies with leucocytes from ten different animal species.

Twenty commercially available monoclonal antibodies (mAbs) raised against various human leucocyte surface antigens were tested on lymphocytes, monocytes and granulocytes from horse, pig, cow, sheep, goat, dog, mink, rabbit, rhesus monkey as well as man. Eight antibodies reacted with leucocytes from some of the animals. These were the antibodies against CD2, CD4, CD5, CD8, CD14, CD18, HLA-DR, and the HLA-ABC antigen. The CD18 antibody reacted with lymphocytes, monocytes and granulocytes from all animals tested except goat. The CD14 antibody reacted with monocytes from pig, cow, sheep, goat, dog, mink, rabbit and monkey and with granulocytes from pig and rabbit. The anti-HLA-ABC reacted with leucocytes from monkey and cow. Finally, the CD2, CD4, CD5 and CD8 antibodies reacted with leucocytes from monkey only.

Improved flow cytometric identification of myelopoiesis by the simultaneous labelling with CD13, CD14 and CD66 monoclonal antibodies.

The aim of the present study was to increase our knowledge of myelopoiesis evaluated by flow cytometry. We therefore designed a triple-marker assay employing monoclonal antibodies against the CD13 (immature), the CD14 (monocytic), and the CD66 (mature myeloid) antigens using three-colour immunofluorescence. In normal donor bone marrow the assay enables simultaneous identification of immature (CD13+, CD14-, CD66-), intermediate (CD13+, myelopoietic differentiation stages through the exclusion of CD14+ monocytic cells. In the diagnosis and longitudinal follow-up of AML patients the assay was of value in the fast determination of remission state. In MDS, the immature myeloid component could be distinguished in patients defined according to the FAB classification with the possibility of identifying aberrant phenotypes, the assay should also be of interest in other myeloproliferative disorders. Moreover, because it is easy to perform, time-saving, and yields comparable results to single antibody reactivity controls, it can replace more tedious and less-informative flow cytometric immunophenotyping procedures.

Response of refractory Hodgkin's disease to monoclonal anti-CD30 immunotoxin.

In Hodgkin's disease, Hodgkin and Reed-Sternberg cells consistently express the antigen CD30. We investigated the possible therapeutic role of an immunotoxin prepared by covalent linking of an anti-CD30 monoclonal antibody (Ber-H2) to saporin (SO6), a type-1 ribosome-inactivating protein. The immunotoxin (0.8 mg/kg in one or two doses) was given to four patients with advanced refractory Hodgkin's disease. In three, there was rapid and substantial reduction in tumour mass (50% to greater than 75%). Clinical responses were transient (6-10 weeks). In-vivo binding of the immunotoxin to tumour cells was shown by immunohistology in two patients. Antibodies to both parts of the immunotoxin developed in all patients.

In vivo targeting of Hodgkin and Reed-Sternberg cells of Hodgkin's disease with monoclonal antibody Ber-H2 (CD30): immunohistological evidence.

The ability of the Ber-H2 (CD30) monoclonal antibody (mAb) to target in vivo Hodgkin (H) and Reed-Sternberg (R-S) cells was investigated in six patients with advanced Hodgkin's disease (HD). The patients were injected with scaled-up quantities of 'cold' Ber-H2 mixed-up to a small dose of 131I-labelled Ber-H2, and in vivo binding of the antibody to H and R-S cells was assessed by immunohistological analysis of tumour biopsies and immunoscintigraphy. Only 50% of tumour sites were imaged at scintigraphy by the 131I-labelled Ber-H2. In contrast, immunohistological studies on tissue biopsies, taken 24-72 h following the mAb injection, showed that H and R-S cells in all tumour sites, including those that were not imaged by immunoscintigraphy, were specifically and strongly labelled in vivo by the injected Ber-H2, at a dose as low as 30-50 mg of antibody. In vivo binding of a single dose of Ber-H2 mAb to H and R-S cells did not result in any anti-tumour effect. The excellent in vivo targeting of H and R-S cells with the Ber-H2 mAb may have been the result of multiple favourable factors, including: (a) the restricted expression of the CD30 antigen in normal human tissues; (b) the low level of soluble CD30 in the serum of our patients; and (c) the high affinity of the Ber-H2 mAb for the CD30 molecule. The immunohistological results presented in this study provide a strong argument for using the Ber-H2 mAb as a carrier for delivering cytotoxic agents (isotopes or toxins) to neoplastic cells of HD refractory to conventional therapy.