A new monoclonal antibody, KP1, raised against a lysosomal fraction of human lung macrophages, recognises a fixation-resistant epitope in a wide variety of tissue macrophages (such as Kupffer cells germinal centre, splenic, and lamina propria macrophages), and in granulocyte precursors. Its broad reactivity with cells of the mononuclear phagocytic lineage was established by testing on routinely processed samples of normal and reactive lymphoid tissues. Interdigitating reticulum cells were unstained or showed limited cytoplasmic staining while Langerhans' cells and follicular dendritic reticulum cells were unreactive. KP1 recognises a molecule of about 110 kilodaltons in macrophage-rich human tissue when tested by either immunoprecipitation or Western blotting (although the latter procedure also shows two additional components with molecular weights of 70 and 40 kilodaltons). KP1 should be of considerable value for studying disorders of the monocyte/macrophage system, including both reactive and neoplastic states (such as true histiocytic proliferations).
The distribution of the pan-macrophage CD68 antigen, recognized by six different monoclonal antibodies, was examined in human blood, tissue, and cell lines using APAAP staining and Western blotting. All antibodies stained monocytes and macrophages, but labelling of neutrophils, basophils, and lymphocytes was seen with some of the reagents. In addition, the CD68 antibodies demonstrated a variety of staining patterns on some non-haemopoietic cells. The subtle differences between the reactions of the different antibodies suggested that the CD68 antigen may be heterogeneous, possibly due to differences in glycosylation. While CD68 antibodies are very useful markers of the macrophage/myeloid series, the presence of small amounts of the antigen on some lymphoid and non-haemopoietic cells means that care should be taken when using them for the diagnosis of tumours of unknown origin.
SUMMARY Three monoclonal antibodies MT1, L60 , and DF-Tl, were reported independently as recognising human T cells in routinely processed, paraffin wax embedded tissue. The present study was performed to compare these three reagents in terms of their immunocytochemical reactions and target molecule(s). On Western blotting of white cell extracts the three antibodies reacted with antigens of the same molecular weight (range 110-160 kilodaltons). Furthermore, their immunocytochemical reactivity with normal human cells, as analysed by two-colour flow cytometry, was essentially identical (labelling ofmonocytes, most T lymphocytes, and weak reactions with some B cells), and the antibodies gave closely similar reactions on 54 white cell derived neoplasms. To identify the target antigen for these three reagents, antibodies from the Third International Workshop on Leucocyte Antigens were reviewed and it was shown that the Western blotting and immunocytochemical reactions of MTl, L60 (Leu-22), and DF-T1 were identical with those of the reagents which defined the CD43 antigen (also known as leucosialin or sialophorin). Furthermore, all these antibodies reacted with cells transfected with a cDNA clone encoding CD43. It is concluded that antibodies MTl, L60 (Leu-22), and DF-T1 all recognise the heavily glycosylated myeloid/lymphoid associated CD43 antigen.There has been much recent interest in monoclonal antibodies which detect fixation resistant antigens and which can be used to distinguish between B and T cell lymphomas in routinely processed paraffin wax sections.' One widely used reagent of this sort is MTI2 which recognises human T cells and myeloid cells. Antibody L60
SUMMARY A tissue processing instrument (the Histokinette) was modified by the addition of an electronic timing device which allows an immunocytochemical staining technique (the APAAP method) to be performed as a semiautomated procedure. After incubation with primary monoclonal antibodies (applied by hand) slides (up to 72 in a batch) are placed in racks and cycled through tanks of reagents, comprising anti-mouse Ig followed by APAAP complexes with intervening timed draining and washing stages. This semiautomated process gave consistent staining results and offered considerable savings in time compared with conventional methods. The same reagent baths were used over four months on an almost daily basis without deterioration in staining intensity, and consequently the calculated overall cost of the staining procedure was less than if the reagents had been applied by hand and then discarded. The machine is now into its eleventh month of operation; the reagents have been changed twice.It is suggested that this approach, because of savings in time and increased consistency, may be an attractive technique for the routine immunocytochemical staining of slides, and that the nature ofthe APAAP method is particularly suitable for automation as the necessary reagents can be produced at low cost.The APAAP immunoalkaline phosphatase technique' (table 1) has been widely used for staining histological and haematological specimens; the advantages of the method have been reviewed elsewhere.2 APAAP staining, however, entails considerable technical time, and results depend, at least in part, on the skill and care of the person performing the staining. There would therefore be considerable benefits in a technique which automated at least part of the procedure. The manual technique is also inherently wasteful in that each of the reagents (usually in a volume of 50-100 p1) is pipetted directly on to the slides and then washed away in buffer at the end of each incubation period. As the antimouse Ig and APAAP are applied at saturating concentration and are not exhausted after a single incubation, there is unnecessary wastage, which would be avoided if the reagents were reused.For these reasons we attempted to develop an automated procedure for performing the APAAP procedure based on incubation of slides in tanks of reagents which can be used repeatedly over a period of time. Incubation with primary antibodies is the most difficult step in the procedure to automate as it is common practice to use several different reagents in a staining run, and primary antibodies are often only
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