Detection of rare malignant cells and their apoptotic fragments in cerebrospinal fluid.

Routine cerebrospinal fluid cytology often fails to detect small numbers of malignant cells exfoliated from primary lymphomas of the central nervous system and metastatic carcinomas. Immunocytochemistry on poly-L-lysine-coated slides was optimised to permit unequivocal identification of a single carcinoma cell in 1 mL of cerebrospinal fluid, as well as carcinoma cell-derived apoptotic bodies that themselves contribute to enhanced diagnostic sensitivity.

Rat monoclonal antibodies differentiating between the Epstein-Barr virus nuclear antigens 2A (EBNA2A) and 2B (EBNA2B).

Rat monoclonal antibodies were produced against the C-terminus of Epstein-Barr virus nuclear antigens 2A (EBNA2A) and 2B (EBNA2B) expressed as bacterial trpE fusion proteins. The initial screening was performed using a soluble bacterial extract containing the fusion proteins. Positive hybridomas were confirmed by immunofluorescence on SF158 (Spodoptera frugiperda) insect cells infected with recombinant baculovirus (Autographa californica nuclear polyhedrosis virus) and expressing the complete EBNA2A or EBNA2B genes. We selected a panel of antibodies which reacted either with both antigens or specifically with EBNA2A or with EBNA2B. The antibodies were extensively characterized using immunoprecipitation, Western blotting, epitope mapping on synthesized peptide segments of EBNA2A, immunocytology, and immunohistology on both cryostat sections and paraffin sections of AIDS-associated primary central nervous system lymphomas.

Optimized detection of cytoplasmic immunoglobulin and CD3 in benign and malignant lymphoid cells: enhanced sensitivity combined with differential staining of endogenous peroxidases and light microscopic morphology.

A novel immunocytochemical approach to the detection of cytoplasmic antigens, exemplified by immunoglobulin and CD3, was evaluated using benign and 155 malignant lymphohematopoietic cell specimens and conventional techniques for detailed comparison. It relies on (a) electrostatic cell attachment to poly-L-lysine-coated multispot slides, (b) sequential use of glutaraldehyde fixation and detergent permeabilization, and (c) staining of endogenous peroxidases and antigens in contrasting colors. Differential staining instead of inactivation of endogenous peroxidases and avoidance of artifacts incurred in conventional cytocentrifugation, dehydration, and alcohol or acetone fixation uniquely afforded improved precision in cell identification, clear distinction of cytoplasmic from surface antigenic staining, and greatly enhanced sensitivity in antigen detection, all combined with increased performance efficiency achieved by the use of multispot slides. With this method, cytoplasmic immunoglobulin and CD3 were found more widely distributed than has been previously recognized. Thus, almost all malignant cells in all cases of B-cell lymphoma/leukemia (encompassing the major subtypes) and T-ALL, as well as substantial proportions of benign mature B- and T-lymphocytes, stained strongly positive for cIg and cCD3, respectively, whereas myeloid cells were consistently negative. High sensitivity combined with excellent cytomorphology and differential myeloperoxidase staining permitted unequivocal differentiation of minute fractions of malignant cells against a heterogeneous background of benign cells.

Immunocytochemical identification of meningeal leukemia and lymphoma: poly-L-lysine-coated slides permit multimarker analysis even with minute cerebrospinal fluid cell specimens.

Use of immunocytology for accurate identification of malignant cells in cerebrospinal fluid (CSF) has so far been hampered by high cell requirements of the immunologic methods hitherto used. In an attempt to minimize cell loss in cytopreparation, electrostatic binding of cells to poly-L-lysine (PLL)-coated multispot slides, followed by immunocytochemistry, was investigated. Using optimized conditions of cell attachment and fixation and performing all washing procedures on the slide made multimarker analysis possible even in paucicellular specimens, while preserving excellent cell morphology and yielding high sensitivity in the detection of antigens. In a study of 26 CSF specimens with inconclusive cytomorphology, comprising 335 single marker determinations, we were able to discriminate reliably between resting or activated benign cells and a wide range of types of malignant lymphoid cell. A definitive diagnosis was reached in all cases by one tap only. Malignant meningitis was ruled out in ten specimens and proved in 16, including five in which the type of malignancy could only be determined by immunophenotyping. We conclude that immunocytochemistry on PLL-coated slides constitutes the method of choice for immunologic cell differentiation in CSF, which allows equivocal morphologic findings to be clarified.

Prethymic phenotype and genotype of pre-T (CD7+/ER-)-cell leukemia and its clinical significance within adult acute lymphoblastic leukemia.

Pretreatment blast cells from 739 adults with acute lymphoblastic leukemia (ALL) were immunophenotyped as part of a prospective treatment protocol study. Among 192 patients (26%) with T lineage ALL, 47 (6%; 24% of T lineage ALL) had lymphoblasts without sheep erythrocyte rosette formation, but with pan-T antigen CD7 on the membrane and intracellular CD3 proteins mostly in perinuclear accumulation. The T-cell surface antigens CD5 and/or CD2 and focal acid phosphatase were additional markers of this subgroup traditionally called pre-T ALL, whereas thymocyte antigen CD1 as well as CD4 and CD8 antigens were not expressed. Hematopoietic progenitor cell markers, namely terminal deoxynucleotidyl transferase (TdT), and in part common ALL antigen (CD10), HLA-DR antigens, and/or My-10 (CD34), a unique antigen of marrow cells absent in thymus cells, further characterized this immature T-ALL form of putative prothymocytic phenotype (CD7+/intracellular CD3+/TdT+/My-10+/-/HLA-DR+/-/CD10+/-). The prethymic T cell character was supported by germ-line T-cell receptor beta genes found in 21 of 36 patients analyzed. In five cases only T gamma-chain genes were rearranged. Fifteen patients, however, had rearrangements of both T beta and T gamma genes. Immunoglobulin heavy chain genes were rearranged only in two cases. Pre-T ALL differed significantly from E-rosette+ T-ALL in some presenting clinical features, namely mediastinal mass, lymphoadenopathy, and platelet count, and independently of clinical factors in prognosis (P = .02, median remission duration: 15.7 v 33.5 months, and P = .02, median survival time: 24.6 v 50.7 months). We conclude that ALL classification based solely on T- or B-cell lineage affiliation is not sufficient but needs further subdivision according to relevant maturation stages as exemplified here within the T-cell axis. The putative prethymic T cell progenitor phenotype described might help elucidate the sequence of genetic events that commit normal hematopoietic cells to the T-cell lineage.

Improved detection of terminal transferase (TdT): the use of detergents on glutaraldehyde-fixed non-dehydrated cells prevents denaturation and diffusion artifacts.

TdT as an intranuclear enzyme mainly of immature lymphoid cells is commonly determined immunologically using air-dried cell smears fixed with methanol. Both cell dehydration and alcohol fixation were found here to denature TdT and surface antigens. This could be prevented by using non-dehydrated cells bound electrostatically to poly-L-lysine-coated slides, fixed minimally with glutaraldehyde and rendered permeable to antibodies by the non-ionic detergent Brij 56. Crosslinking glutaraldehyde in addition prevented diffusion of TdT to extranuclear sites. By avoiding artifacts of denaturation and diffusion, a higher sensitivity in the detection of TdT was achieved despite considerably lower quantities of antibody.