Haim Gamliel

Air-drying of human leucocytes for scanning electron microscopy using the GTGO procedure

The utilization of tannic acid and guanidine hydrochloride as mordants for better osmium binding has been shown to serve as an excellent alternative to metal coating of organ tissue specimens for scanning electron microscopy (SEM). The present report describes the GTGO procedure, a modification of the TAO technique introduced by Murakami et al. (1977, 1978), which we have found successful for the preparation of air dried peripheral blood leucocytes for SEM studies. Air dried, GTGO‐treated leucocytes show excellent preservation of surface features with minimal cell shrinkage. When critical point dried, GTGO‐treated cells are examined, they also show less shrinkage than cells prepared with standard glutaraldehyde fixation and critical point drying. The potential application of this air drying procedure (GTGO‐AD) to other soft biological specimens is currently under investigation. This technique is recommended as a new and effective air drying procedure for the successful preparation of cells for SEM.

Surface morphology and membrane phenotype of cultured human leukemia-lymphoma cells: A scanning electron microscopic study of 36 cell lines

Scanning electron microscopy and immunologic methods, to detect the expression of a variety of surface markers, were performed on cells from 36 established human leukemia‐lymphoid cell lines. Attempts were made to correlate the surface morphologic findings with the membrane phenotype as determined by the presence or absence of a number of specific antigens and B‐ or T‐cell markers. Thirteen of the cell lines were of the T‐lymphoid type, 15 B‐derived, and eight were defined as non‐B non‐T in nature. All the lines derived from patients with acute lymphoblastic leukemia (ALL) had similar surface topographies and generally displayed relatively smooth surfaces with few microvilli, while in some a proportion of moderately villous cells was evident. Burkitts lymphoma cells tended to show more villous surfaces but, similar to circulating B‐ALL cells, variable numbers of microvilli were frequently seen making consistent distinctions between this and other lymphoid leukemias difficult in individual cases. Two of the non‐B non‐T lines are known to be of erythroid (K‐562) and myeloid origin (HL‐60), respectively. In both these lines, cells with relatively few microprojections dominated; however, some showed transverse ridge‐like profiles, a feature frequently encountered on circulating leukemic cells of myeloid type.

Burkitt's lymphoma cells: Membrane properties and surface morphology as seen by scanning electron microscopy

Abstract Membrane properties and surface morphology were studied in cells from 13 different cultured Burkitts lymphoma cell lines in an attempt to determine whether there was any structure-function correlation for these cells. Burkitts lymphoma cells were all bone marrow (B)-derived and most were spherical in shape with moderate to markedly villous surfaces. There was no obvious correlation between the number of surface microvilli seen under the scanning electron microscope and the degree of expression of the different B-cell surface markers and other membrane properties.

Simultaneous Presentation of Plasma Cell and Monocytic Leukemia with a Subacute Clinical Course

A rare case of simultaneous presentation of monocytic and plasma cell leukemia is reported. Cytochemistry, transmission and scanning electron microscopy confirmed the presence of a dual population consisting of monoblasts and plasma cells. Monoblasts contained nonspecific esterases, secreted lysozyme and showed dense bodies and surface ruffles under the scanning electron microscope, while the plasma cells secreted IgG kappa paraprotein, contained rough endoplasmic reticulum, and showed surface blebs with microvilli. Another unusual feature of this case was the relatively chronic course of the disease, lasting 15 months after initial diagnosis.

Scanning immuno‐electron microscopy of human leukaemia and lymphoma cells: a comparative study of techniques using immunolatex spheres as markers

In this study scanning immuno‐electron microscopic (SIEM) techniques were used to identify human leukaemia‐lymphoma cells. Monodispersed polystyrene (latex) beads were conjugated to specific antisera using glutaraldehyde, in an attempt to detect surface antigenic components on a variety of cells of known origin. Antisera, mostly immunoglobulin fractions, against human thymus (T) derived cells, common type acute lymphoblastic leukaemia cells (C/ALL) and surface immunoglobulin (sIg) bearing cells were used to coat latex spheres, while rabbit anti‐mouse Thy‐1 antiserum or whole human‐IgG (γ‐globulin) bound to latex were used as controls in some experiments. The use of SIEM techniques in the direct mode as a simple and sensitive method for labelling surface antigens is described. The disadvantages of the SIEM methodology are also summarized while the requirements for optimal cell preparation using this technique are stressed. The experiments were designed to ascertain whether prolonged fixation of cells could be used prior to incubation of the cells with the marker. In this respect, repeated neutralization of the glutaraldehyde with glycine is essential. SIEM labelling of cells is random and unreliable without adequate quenching with glycine. The heteroantisera used in this study proved to be adequate and insignificant non‐specific attachment and cross reactivity were seen. SIEM adds a further dimension to ultrastructural aspects of immunology and is a potentially useful tool in the study and identification of leukaemia and lymphoma cells.

Prolymphocytic leukaemia: surface morphology in 21 cases as seen by scanning electron microscopy and comparison with B-type CLL and CLL in ‘prolymphocytoid’ transformation

Summary. The surface architecture of leukaemic cells obtained from 21 cases of proven prolymphocytic leukaemia (PLL) and eight cases of chronic lymphocytic leukaemia (CLL) with ‘prolymphocytoid’ transformation (PL‐CLL) was compared with the cell surface morphology of leukaemic cells obtained from 46 cases of B‐type CLL, using the scanning electron microscope (SEM). All cases were defined by cytochemistry, immunological markers and transmission electron microscopy prior to SEM examination. B‐CLL cells showed the well‐recognized spectrum of surface architecture described in earlier studies. The majority of cells had moderate numbers of short microvilli, although in a minority, cells with relatively smooth surfaces predominated. In seven of the eight cases of PL‐CLL, cells were villous in nature and in this respect similar to CLL cells; however, more cells with dense microvilli were seen. The prolymphocytic cells were recognized by their larger size and in 18 of the 19 cases of B‐derived PLL, villous cells predominated. Two cases of T‐derived PLL showed variable cell surface morphology ranging from smooth to moderately villous. It appears that B‐PLL cells are most frequently villous and display more surface microvilli than B‐CLL cells. B‐prolymphocytes display the surface features regarded as characteristic for neoplastic B‐cells as seen in patients with B‐type lymphoma and leukaemia.

Utilization of monoclonal antibodies and immuno-scanning electron microscopy for the positive identification of human leukemic cells

Monoclonal antibodies generated against normal and leukemic human leukocytes were tested for their differential reactivity with leukemia and lymphoma cell lines as well as with circulating lymphoid and myeloid leukemic cells by means of immuno-scanning electron microscopy (immuno-SEM). Anti-T (OKT3), anti-μ-chain, anti-CALLA (J5), anti-BA-1, anti-BA-2, and anti-nonlymphoid (Mol) monoclonal antibodies were covalently conjugated to polystyrene latex microspheres (immunolatex), using a two-step glutaraldehyde reaction, and subsequently incubated with the various cell types. Cultured B-type Burkitt lymphoma cells (Daudi) and chronic lymphocytic leukemia (CLL) cells displayed extensive labeling with monoclonal anti-μ, anti-B1, and anti-BA-1 immunolatex conjugates, while cultured malignant T cells (HD-Mar) showed positive labeling with OKT3 immunolatex alone. Cultured myelomonocytic cells (GDM-1) and cells obtained from patients with acute myeloblastic (AML) and monoblastic leukemia (AMoL) labeled only with anti-Mol immunolatex, while cultured promyelocytic cells (HL-60) displayed far less labeling with this conjugate. Commontype acute lymphoblastic leukemia (C/ALL) cells were labeled predominantly with the J5 (anti-CALLA) and anti-BA-2 immunolatex conjugates. Evidence is presented indicating that immuno-SEM employing monoclonal antibodies is a reproducible technique which may be used in the study of leukocyte maturation and may provide additional information in the classification of poorly differentiated leukemias.