Rachel Leizerowitz

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.

Establishment and characterization of a new permanent cell line (GDM-1) from a patient with myelomonoblastic leukemia☆

The GDM-1 permanent cell line was established from the peripheral blood of a patient with a Philadelphia chromosome negative myeloproliferative disorder, after transformation to acute myelomonoblastic leukemia. The GDM-1 cells exhibited the same characteristics as those isolated from the peripheral blood of the patient prior to death: cells contained non-specific esterase sensitive to fluoride, myeloperoxidase, lysozyme (muramidase), and exhibited both Fc and complement (C3) receptors but lacked B- and T-cell surface markers including T-associated antigens. E-rosetting capacity, surface and intracytoplasmic immunoglobulins and EBV determined nuclear antigen (EBNA). The GDM-1 cells bore the 1a receptor and the myeloid leukemia antigen (M-1). The karyotype of the cultured leukemic cells showed the same specific chromosomal abnormalities present in the monoblasts obtained from the peripheral blood prior to death, indicating that the cell line was derived from the original leukemic cells.

“Jumping translocation” in a 17-month-old child with mixed-lineage leukemia☆

A 17-month-old child with acute biphenotypic (pre B-ALL/myelomonocytic) leukemia is reported. Extensive cytogenetic analysis performed at various stages of the disease revealed a clonal evolution at the time of initial diagnosis with two types of abnormal clones, one with trisomy 22 and two other related clones with trisomy 22 plus partial trisomy of the long arm of chromosome 1 associated with the telomeric segment of either chromosome 20q or 21p. At the time of relapse the only abnormal clone involved trisomy 22 and partial trisomy of 1q, but this time in association with the telomeric segment of 14p. The unique feature of these translocations is discussed and the possibility of the correlation between the different chromosomal abnormalities and the expression of biphenotypic markers is raised.

Farage, a Novel Early B Cell Lymphoma Cell Line with Trisomy 11

Farage, a new cell line established from a lymph node biopsy of a patient with non-Hodgkins lymphoma (NHL), constitutes a clonal expansion of cells at a distinct stage of B-cell differentiation. The cells lack both T and myeloid surface markers, express B cell surface antigens including CD19, CD20, CD22, HLA-DR, were positive for C3 receptors and EBNA and expressed BCL-2. No immunoglobulin determinants could be demonstrated on the cell surface. Intracellular IgM and kappa chains were detected, in an unusual but distinct localization and appeared to be localized to the nucleus or to the perinuclear area without any spread to the cytoplasm, as seen in the early B cells. Southern blot DNA analysis showed rearrangement of one of the IgJH alleles. The Farage cells were negative for B cell activation antigens including CD25, CD11b, HC2 and Bly-7. The cells were negative for two anti CD-10 (CALLA) reagents but weakly positive with one. Interestingly they were strongly positive for both nuclear and cytoplasmic T...

Myelomonocytic Antigens are Rarely Expressed on B-Lymphocytic Leukemia Cells

In the light of recent observations reporting that B-lymphocytic leukemia (B-CLL) cells may express a variety of myelomonocytic antigens, 28 patients with B-CLL and B-leukemic lymphocytic lymphoma were studied for the presence of these antigens using monoclonal antibodies to detect CD13, CD33, CD15 and CD14. Analysis of immunofluorescence (IF) was carried out by two procedures; one which employed the standard conventional method of gating used in our laboratory for flow cytometry, while the other procedure increased the sensitivity of the analysis, by moving the marker for IF to the left, so as to widen the gate to include more cells with low IF. Using the conventional methodology, the mean proportion of cells considered positive was less than 3% for any of the 4 markers studied. In only a few patients were 5% or more of the B-CLL cells positive for some of the markers studied (3 patients with 6.2-11.3% CD13+; 2 with 6.0-9.6% CD14+, and one with 11.8% CD15+ cells). No case had more than 2.5% + CD33+ cells. The second procedure with a wider gate to enhance sensitivity for less positive cells, increased the number of positive cells for any of the markers in only 4 patients. These results are contradictory to others reported recently, and some of the possible causes for this discrepancy are discussed. It is suggested that more useful data may be obtained if the level of staining intensity and patterns of positive staining are documented in the future.

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.

Expression of CD11a (LFA-1) on B-chronic Lymphocytic Leukemia and Lymphoma Cells: Correlation with Cell Surface Immunoglobulin Intensity and CD58 (LFA-3) Expression

In the present study the expression of CD11a (LFA-1) was studied on B-chronic lymphocytic leukemia (CLL) cells and B-non-Hodgkins small lymphocytic lymphoma in leukemic phase (NHL). The expression of CD11a was correlated with that of surface immunoglobulin (Smig) and CD58 (LFA-3). Patients with CLL were found to have a significantly lower proportion of CD11a+ cells than NHL patients, and the intensity of the staining of Smig was lower in CLL when compared with NHL. The proportion of CD11a+ cells in CLL, but not in NHL, was inversely correlated with the total white blood cell count. In CLL patients the percentage of CD11a+ cells was significantly lower in Rai stages 2-4 compared with stages 0-1. There was a strong correlation between the proportion of CD11a+ and CD58+ cells in both CLL and NHL. In contrast, there was no correlation between the proportion of CD11a cells and Smig intensity in both of these diseases when studied separately. However, when the results in these two diseases were pooled, the proportion of CD11a cells correlated with Smig intensity. The present study indicates that Smig intensity and proportion of CD11a+ cells are important criteria for the differential diagnosis between CLL and NHL.

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.

In vitro differentiation and establishment of cell lines derived from human myelomonocytic leukemia cells

Primary cultures of cells derived from 13 patients with acute myelomonocytic leukemia (AMML) were studied with particular emphasis on in vitro proliferation, cell differentiation and the mode for establishment of cell lines. Using irradiated human macrophage monolayers to assist cell growth, we obtained four new cell lines of myelomonocytic origin. All the cell lines were characterized for cytochemical markers and response to phorbol esters (TPA), a differentiation inducing agent. In the absence of any inducing agent, spontaneous differentiation of blast cells into mature macrophages-like cells occurred in 8 out of the 13 primary cultures. Thus, maturation induction by agents such as TPA is not always required in order to obtain leukemic cell differentiation in vitro. The regulation of cell proliferation and differentiation by cellular interactions and by extrinsic soluble products is discussed in detail, in the light of these findings.

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.