A Modified Cytochemical Method for DAP IV Demonstration which Enables Simultaneous Visualization of Cell Surface Immunostaining

Dr. Antonino Carbone, Division of Pathology, Centro di Riferimento Oncologico, Via Pedemontana Occidentale, I-33081 Aviano (Italy) The cytochemical demonstration of dipeptidyl amino peptidase IV (DAP IV) in human peripheral blood lymphocytes was first described by Lojda [1]. Subsequent reports have shown that this ‘exopepti-dase’ has an apparent restriction to a particular T-cell subset, in that most Tlymphocytes bearing receptors for IgM (Tμ cells) display DAP IV reactivity [2]. Crockard et al. [3], by using a combined monoclonal antibody/immunocolloidal gold technique and cytochemical method for DAP IV reactivity, demonstrated that single or several granules of DAP IV reaction products were present in 72% of OKT3+ and OKT4+ cells, whereas a significantly lower percentage of OKT8+ cells displayed positivity. Recently, a modified cytochemical method was used to show DAP IV in peripheral blood buffy coat preparations and bone marrow smears in both normal and malignant hemic cells [4]. More recently, a modified histo-chemical method was used to show the presence of DAP IV in fixed, freeze-dried, cryostat sections of reactive and neoplastic lymphoid tissues [5]. The value of this method in the cytological classification of lymphomas was emphasized [5]. We would like to report technical results of a work undertaken in our laboratories in order to ascertain the optimal conditions for showing cytochemically the presence of DAP IV in lymphocyte subsets defined simultaneously by monoclonal antibodies in cytospin preparations from cell suspensions. Tonsillar and lymphoid tissues were minced and passed through a metal mesh (200–400 μm diameter). The cells were resuspended in RPMI 1640 (Flow Lab. Inc.) with the addition of fetal calf serum at 5% (Flow Lab. Inc.); then they were washed in the same me1 This work was supported in part by a grant from the Associa-zione Italiana per la Ricerca sul Cancro, Milan, Italy. dium. Mononuclear cells were isolated from hepari-nized blood samples from healthy donors on Ficoll-Isopaque (Pharmacia) density gradient. From blood, tonsils, and lymph nodes, cytospin slides were prepared. The cytochemical demonstration of DAP IV was performed according to the method of Lojda et al. [6] with slight modifications (table I); these changes permitted good simultaneous visualization of the final reaction products of an immunocytochemical method combined with enzymocytochemistry. The method for demonstrating DAP IV activity was performed as follows: the slides were fixed 5 min in acetone; 0.4 mg/ml glycyl-prolyl-4-