Scanning electron microscope cytochemistry of normal and leukaemic leukocytes

Backscattered Electron Imaging (BEI) is a particular technique which permits to study cytochemical reactions with the Scanning Electron Microscope (SEM). The BEI data pertaining to specific enzymatic activities can be directly correlated to the surface morphology of each individual cell. Leukocytes from 5 normal individuals, 14 patients with acute nonlymphoblastic leukaemia (ANLL), 7 patients with chronic myeloid leukaemia (CML) and 3 patients with acute lymphoblastic leukaemia (ALL) were studied for myeloperoxidase activity, acid phosphatase localization, silver staining of the nuclei and phagocytosis of iron carbonyl in the BEI mode of SEM. Some normal peripheral blood leukocytes which cannot be distinguished by their surface morphology alone were satisfactorily identified with the BEI technique. Leukaemic myeloid cells can be recognized in many cases because of their positive myeloperoxidase reaction, while monocytic elements can be characterized by the presence of surface ruffles, acid phosphatase activity and active phagocytosis. The usefulness of the BEI technique in identifying different blood cell types with the SEM and its possible application to the diagnosis of certain cases of leukaemia are discussed.     

Ultrastructural cytochemical localizations by back-scattered electron imaging of white blood cells

White blood cells have been studied in the back-scattered electron imaging (BEI) mode of scanning electron microscopy (SEM) with cytochemical methods for endogenous peroxidase, acid phosphatase, and a silver-staining method for nuclei. Peroxidase-positive granules were seen with good contrast and resolution in myeloid precursor cells and acid phosphatase activity was easily detected in macrophages and monoblasts. Silver staining permitted recognition of the shapes and location of the nuclei. In spite of the cytochemical procedures, cell surface structures were reasonably well-preserved in all methods, making direct correlation of BEI and secondary electron imaging (SEI) images an attractive feature in cell research with the scanning electron microscope.     

Distribution pattern of acid phosphatase activity in human peripheral blood leukocytes: a cytochemical scanning electron microscopy study

The distribution patterns of acid phosphatase hydrolytic activity were studied in human peripheral blood cells with enzymocytochemical techniques together with light and scanning electron microscopy in the secondary and backscattered electron imaging modes. The acid phosphatase reaction product was seen in three different patterns of distribution: focal, granular and diffuse. These patterns were correlated with similar findings obtained with light microscopy. Acid phosphatase distribution patterns seen with SEM in the BEI mode were also correlated with the surface morphology of peripheral blood cells seen in the SEI mode. Cells exhibiting the focal pattern were smooth-surfaced with few microvilli; cells showing a granular pattern presented microvilli and microridges; ruffles were characteristic of cells with a diffuse pattern of activity. No reaction product was seen in cells bearing microvilli or ridges. Our findings demonstrate the correlation between acid phosphatase activity patterns and surface features in different subpopulations of peripheral blood cells.     

Distribution pattern of acid phosphatase activity in human peripheral blood leukocytes: a cytochemical scanning electron microscopy study

Summary The distribution patterns of acid phosphatase hydrolytic activity were studied in human peripheral blood cells with enzymocytochemical techniques together with light and scanning electron microscopy in the secondary and backscattered electron imaging modes. The acid phosphatase reaction product was seen in three different patterns of distribution: focal, granular and diffuse. These patterns were correlated with similar findings obtained with light microscopy. Acid phosphatase distribution patterns seen with SEM in the BEI mode were also correlated with the surface morphology of peripheral blood cells seen in the SEI mode. Cells exhibiting the focal pattern were smooth-surfaced with few microvilli; cells showing a granular pattern presented microvilli and microridges; ruffles were characteristic of cells with a diffuse pattern of activity. No reaction product was seen in cells bearing microvilli or ridges. Our findings demonstrate the correlation between acid phosphatase activity patterns and surface features in different subpopulations of peripheral blood cells.     

T-cell-antigen positive, E-rosette negative acute lymphoblastic leukaemia (author's transl)]

The lymphoblasts from 100 patients with acute lymphocytic leukaemia were investigated for the expression of receptors for sheep erythrocytes (E) and of a specific heterologous T cell antigen (T). In 17 cases, both T cell markers were expressed simultaneously on the leukaemic cells. In 13 cases only T antigens could be demonstrated on the lymphoblasts. A quantitative analysis of T antigens by immunoautoradiography revealed that the T expression of E-T+ -lymphoblasts was in general like that of E+T+-lymphocytes in the blood of normal persons, in several cases even higher. Therefore, the failure of E-rosette formation cannot be correlated to a decrease of the other T cell differentiation marker. In 7 out of 9 tested cases, a strong acid phosphatase reaction product located paranuclearly could be demonstrated. Complement-receptors were expressed in 3 of 5 cases which were also demonstrated in some cases of the E+T+-ALL group. The latter group was characterized by a T antigen expression like that of thymocytes. 4 cases of the E-T+ALL group were adults. Since the leukaemia cells of 2 cases were negative for acid phosphatase, PAS and all surface markers including cALL antigen, the T antigen can classify undifferentiated and otherwise unclassificable leukaemias. The clinical signigicance of the E-T+-ALL seems to be important since 5 out of 9 children with this type of ALL died soon after diagnosis.     

Quantitative cytochemistry of blood neutrophils in myelodysplastic syndromes and chronic granulocytic leukaemia

Quantitative cytochemistry of components of blood neutrophil azurophilic granules (myeloperoxidase, chloroacetate esterase, beta-glucuronidase, and acid phosphatase) and specific granules (lactoferrin) has been performed by scanning and integrating microdensitometry in 13 patients with a myelodysplastic syndrome and 11 patients with chronic granulocytic leukaemia. Both patient groups showed a reduction of enzyme activity in azurophilic granules, and also of lactoferrin, consistent with abnormal development of neutrophil granules. These cytochemical changes in blood neutrophils are similar to those found in acute myeloid leukaemia, are consistent with a leukaemic maturation defect, and may be of diagnostic value.     

Cytochemistry of leukocytes in children. IV. The use of cytochemical tests in the differentiation and prognostic evaluation of acute leukemias]

From 63 children with acute leukaemia the bone-marrow smears were cytochemically examined before the beginning of therapy. The activity of peroxydase was examined according to Sato and Sekya, that of acid phosphatase according to L?ffler and Berghoff, that of alpha-naphthyl-acetate-esterase according to Gomori; the evidence of glycogen was examined by means of the PAS-diastase response according to McManus. Among the 63 cases of leukaemia we found 6 cases of paramyeloblastic leukaemia, 2 cases of parapromyelocytic leukaemia, and 3 cases of myelomonocytic leukaemia. 52 cases of leukaemia could not be further differentiated in morphological respect. They represented an immature paraleukoblastic leukaemia. A division according to leading cytochemical criteria was made for them. The therapeutic possibility of influencing the various groups was checked by means of prolonged observations. Children affected with paraleukoblastic leukaemia of the phosphatase type had a significantly low rate of remission similar to the myeloid leukaemia. Paraleukoblastic leukaemia of the PAS type, esterase type and the undifferentiated type revealed no essential differences. The rate of remission, however, was highest in leukaemia of the PAS type amounting to 100%. In one part of patients the prolonged cytochemical observations in 8 children with recidives showed that the cytochemical type under chemotherapy was changed.     

Serological detection of B-cell ("Ia-like") antigens in serum of normal donor and in some sera of leukaemic patients

Some sera from normal donors (1/18) and from leukaemic patients (2/7 with acute myeloid leukaemia [AML], 1/4 with chronic lymphatic leukaemia [CLL], 0/3 with acute lymphoblastic leukaemia [ALL]; with high numbers of leukaemic cells expressing Ia-like p28,33 antigen on the leukaemic cell surface) inhibited the complement mediated cytotoxic activity of highly specific xenogenous anti Ia-like sera (which were prepared by immunization of rabbits with insoluble membrane fractions of B-type lymphoid lines) at a titre 1:4 or less. This effect was not observed with antisera directed against other membrane marker determinants (e.g. T lymphocyte specific antigens). These results suggest that at least a small proportion of membrane bound Ia-like antigens can be released from cell surfaces and in some patients these Ia-like moieties are detectable (by sensitive inhibition assays) in the serum.     

The nature of blast cells in myelodysplastic syndromes evolving to acute leukaemia

The blast cells from nine patients with an overt acute leukaemia following a previous myelodysplastic syndrome (MDS) are analyzed with a panel of monoclonal antibodies as well as by morphological and cytochemical criteria. By integrating the results obtained with these three approaches the leukaemia in 6 patients was assessed as myeloid-granulocytic and/or monocytic-, in two as mixed- megakaryoblastic/myeloid- and in one as lymphoid. A good correlation between morphology, cytochemistry and immunological markers was observed in 7 out of the 9 cases. In three cases a noteworthy percentage of J5+ cells was detected. The exceptional finding of lymphoid as well as megakaryocytic and myeloid transformations suggests that the target cell for these leukaemias could be a pluripotent stem cell.     

In vitro cell adhesion and integrin expression by calcium ionophore-treated mononuclear cells from patients with acute myeloid leukemia

As shown elsewhere, cultured acute myeloid leukaemia blasts acquire certain characteristics of dendritic cells upon stimulation with cytokines and calcium ionophore. The ability of leukaemia-derived dendritic-like cells to express immune costimulatory molecules and dendritic cell marker CD83 has been extensively investigated. Although migratory capacity is a major attribute of dendritic cells, the ability of in vitro modified blasts for adhesion, chemotaxis and homing remain elusive. In the present paper, we show that after stimulation with calcium ionophore acute myeloid leukaemia blasts as well as normal dendritic cell precursors demonstrate increased capacity of binding fibronectin and denatured collagen. The expression pattern of integrins on dendritic-like leukaemic cells in general closely resembles that of monocyte-derived dendritic cells, however, variation in cell properties isolated from blood of individual patients are observed.     

Bacterial glutaminase in treatment of acute leukaemia.

A glutaminase-asparaginase enzyme from Achromobacter sp has antitumour activity in vitro and in animals. Glutaminase was administered in doses of 3500-20 000 IU/m2 body surface area/day to six patients with acute lymphoblastic leukaemia (ALL) and three patients with acute myeloid leukaemia (AML). The enzyme had a blood half life of 80 minutes but depletion of blood glutamine persisted for 12 hours after single doses. Seven patients, including four (two with AML and two with ALL) resistant to asparaginase, received repeated doses of glutaminase. Antileukaemic effects were observed in all seven; one elderly patient developed metabolic acidosis. Study of this new antileukaemic agent in patients with acute leukaemia at an earlier stage of their disease is now justified.     

Humoral detection of leukaemia-associated antigens in presentation acute myeloid leukaemia

The serological analysis of recombinant cDNA expression libraries (SEREX) technique was used to immunoscreen a testes cDNA expression library with sera from newly diagnosed acute myeloid leukaemia (AML) patients. We used a testis cDNA library to aid our identification of cancer-testis (CT) antigens. We identified 44 antigens which we further immunoscreened with sera from AML, chronic myeloid leukaemia (CML), and normal donors. Eight antigens were solely recognised by patient sera including the recently described CT antigen, PASD1, and the cancer-related SSX2 interacting protein, SSX2IP. RT-PCR analysis indicated that we had identified three antigens which were expressed in patient bone marrow (BM) and peripheral blood (PB) but not in normal donor samples (PASD1, SSX2IP, and GRINL1A). Real-time PCR (RQ-PCR) confirmed the restricted expression of PASD1 in normal donor organs. Antigen presentation assays using monocyte-derived dendritic cells (mo-DCs) showed that PASD1 could stimulate autologous T-cell responses, suggesting that PASD1 could be a promising target for future immunotherapy clinical trials.     

Plasma transcobalamins in haematological disorders

Plasma UBBC-B12 and transcobalamins were measured in 112 patients suffering from different haematological disorders. The data showed different patterns of changes in plasma transcobalamin profile in different haematological disorders. Plasma UBBC-B12 and transcobalamins were significantly higher than normal in untreated chronic myeloid leukaemia, acute promyelocytic leukaemia, nutritional megaloblastic anaemia and in refractory anaemias with hypercellular marrow. Normal levels of these proteins were noted in chronic lymphatic leukaemias, in primary and secondary hypereosinophilic states and in multiple myeloma. Subnormal levels of these proteins were observed in hypoplastic anaemia and acute lymphoblastic leukaemia. Chronic myeloid leukaemia patients during blast crisis and acute myeloid leukaemia patients except those suffering from acute promyelocytic leukaemia showed varying pattern of plasma transcobalamins depending on type of blast crisis or FAB subtype of AML. The significance of these changes in plasma transcobalamins have been discussed along with the experience of other workers in this field.     

Decreasedin vitro chemosensitivity of tumour cells in patients suffering from malignant diseases with a poor prognosis

The aim of the study was to assess the predictive value of MTTin vitro assay for evaluation of tumour cell resistance/sensitivity to cytotoxic drugs. We analyzed 105 samples of malignant cells of different origin. The study included patients with a diagnosis of acute and chronic lymphatic leukaemia, acute and chronic myeloid leukaemia, non-Hodgkin lymphoma, carcinoma of the lung, stomach and liver, rhabdomyosarcoma and breast carcinoma. The results demonstrate outstanding chemosensitivity in the majority of childhood acute lymphoblastic leukaemias, medium chemosensitivity of adult haematopoietic malignant diseases and chemoresistance of solid tumour cells. Our preliminary data suggest a good correlation betweenin vitro MTT assay and clinical curability of individual malignant diseases.Abbreviations ALL acute lymphoid leukaemia - AML acute myeloid leukaemia - CML chronic myeloid leukaemia - LCS₅₀ 50% leukaemic cell survival     

Suppression of the in vivo malignancy and in vitro cell multiplication of myeloid leukemic cells by hybridization with normal macrophages.

Mouse myeloid leukemic cells that multiplied in vitro and were malignant in vivo were hybridized with non-malignant non-multiplying normal mouse or human macrophages. Both the hybrids with mouse and with human macrophages were non-malignant and non-multiplying. The suppression of malignancy and cell multiplication in these hybrids was associated with the expression of eight other properties expressed in normal macrophages but not in the myeloid leukemic cells. These properties were C3 and Fc rosettes and immune phagocytosis, rosettes and phagocytosis of uncoated erythrocytes, synthesis and secretion of lysozyme and a high frequency of cap formation by concanavalin A (ConA). The hybrids also expressed two properties of the myeloid leukemic cells, a lack of cell attachment to the surface of a Petri dish and staining for myeloperoxidase. The use of specific antibodies has shown that the lysozyme produced by hybrids between human macrophages and mouse myeloid leukemic cells was human lysozyme. The results indicate that the in vivo malignancy and in vitro cell multiplication of myeloid leukemic cells was suppressed in hybrids with normal macrophages, and that his suppression can be dissociated from the normal macrophage property of cell attachment.     

Routine use of backscattered electron imaging to visualize cytochemical and autoradiographic reactions in semi-thin plastic sections.

The scanning electron microscope (SEM) was used to examine cytochemical and autoradiographic reactions in 2-microns semi-thin sections of tissues conventionally fixed and embedded in various resins. The sections were examined using both the secondary and backscatter modes of the SEM at magnifications within the range attainable with the light microscope. Both modes allowed the imaging of phosphatase reaction product using cerium and lead capture, lectin-gold, and immunogold labeling, with and without silver enhancement, and autoradiography. Backscattered electron imaging (BEI), however, provided images with more contrast and structural details. This approach allows examination of large sections, with more contrast and resolution than the light microscope, and visualization of reactions not visible with this instrument. The improved imaging and the simple and conventional preparation of specimens indicate that BEI can be used routinely to examine tissue organization, cell structure, and the content of the various cell compartments with a resolution approaching that of transmission electron microscopy.     

Fibronectin and factor VIII-related antigen in acute leukaemia

The glycoprotein fibronectin is, as well as by various other cells, also produced in leucocytes and is said to play an important role in malignant transformation of cells. Therefore, the behaviour of plasma fibronectin and of factor VIII R:AG was investigated in acute leukaemia in order to prove their significance as prognostic and therapeutic markers (method: electroimmunoassay). In patients with acute myeloid leukaemia (n = 29) and acute lymphoblastic leukaemia (n = 11) no significant changes in fibronectin concentration could be evaluated. Fibronectin levels declined significantly only during therapy with asparaginase in patients with acute lymphoblastic leukaemia, probably as a result of disturbed synthesis in the liver. Using crossed immunoelectrophoresis against fibronectin antiserum, one normal and one slower migrating antigen (FN:C) could be observed in nearly all plasma samples in patients with acute leukaemia. By means of in vitro tests with highly purified substances and intermediate gel electrophoresis it could be shown that FN:C represents fibronectin which has bound fibrinogen, probably crosslinked by activated factor XIII. Factor VIII R:AG was found to be greatly raised in patients with acute leukaemia--up to 1400% of the normal level. Increased levels correlated well with a worsening of the disease. The protein seems to be suitable for estimating the activity and prognosis of acute leukaemia.     

Mapping gold-labeled IgE receptors on mast cells by scanning electron microscopy: receptor distributions revealed by silver enhancement, backscattered electron imaging, and digital image analysis

Immunogold labeling and silver enhancement techniques are widely used to determine density and distribution of cell membrane receptors by light and transmission electron microscopy. However, these techniques have not been widely used for receptor detection by scanning electron microscopy. We used antigen- or protein A-conjugated colloidal gold particles, together with silver enhancement, sequential secondary and back-scattered electron imaging (SEI and BEI), and digital image processing, to explore cell surface distribution of IgE-receptor complexes on RBL-2H3 cells, a rat leukemia line that provides a model for the study of mucosal mast cells. Cells were first incubated with a monoclonal antidinitrophenol IgE (anti-DNP-IgE) that binds with high affinity to cell surface IgE receptors. The resulting IgE-receptor complexes were cross-linked either with the multivalent antigen, DNP-BSA-gold, or with a polyclonal anti-IgE antibody. Antibody-treated cells were labeled after fixation with protein A-gold. Fixed, gold-labeled cell monolayers were silver enhanced (or not), dehydrated, critical point-dried, and coated with gold-palladium (for SEI analysis) or carbon (for combined SEI/BEI analysis). They were observed in an Hitachi S800 SEM equipped with a field emission tip and a Robinson backscattered electron detector. An image processor (MegaVision 1024XM) digitized images directly from the S800 microscope at 500-1000 line resolution. Silver enhancement significantly improves detection of gold particles in both SEI and BEI modes of SEM. On gold-palladium-coated samples, 20-nm particles are resolved by SEI after enhancement. BEI resolves 15-nm particles without enhancement and 5- or 10-nm particles are resolved by BEI on silver-enhanced, carbon-coated samples. Neither BEI nor SEI alone can yield high resolution topographical maps of receptor distribution (BEI forms images on the basis of atomic number contrast which reveals gold but not surface features). Image analysis techniques were therefore introduced to digitize, enhance, and process BEI and SEI images of the same field of view. The resulting high-contrast, high-resolution images were superimposed, yielding well-resolved maps of the distribution of antigen-IgE-receptor complexes on the surface of RBL-2H3 mast cells. The maps are stored in digital form, as required for computer-based quantitative morphometric analyses. These techniques of silver enhancement, combined BEI/SEI imaging, and digital image analysis can be applied to analyze density and distribution of any gold-labeled ligand on its target cell.     

Leukocyte lysozymes in various forms of leukemia]

The amount of lysozyme in the leukocytes of 47 patients with different forms of leukaemia and 6 healthy persons was investigated. The lysozyme determination was carried out in the lysate of isolated leukocytes obtained after freezing and thawing it seven times. The results expressed in microgram per 10(6) cells were compared with the simultaneously determined lysozyme concentration of serum and urine. A substantial reduction of the lysozyme amount as compared with the normal value (3.1 microgram/10(6) cells) was determined in the leukocytes of patients suffering from chronic lymphatic leukaemia, acute lymphatic leukaemia and the blastic crisis of chronic myeloid leukaemia. Different amounts of lysozyme ranging from extremely low ones to strongly elevated ones were found in leukocytes taken from patients with acute myeloblastic and chronic monocytic leukaemia. In many cases there was a lack of correlation between the lysozyme content of leukocytes on the one hand and that of serum and urine on the other hand. Possible causes underlying this lack of correlation are discussed.     

Preferential induction of apoptosis of leukaemic cells by farnesol

Farnesol preferentially inhibits proliferation and induces apoptosis of tumour-derived but not non-transformed cell lines. We investigated whether farnesol induces apoptosis of blasts from patients with acute myeloid leukaemia (AML) and leukaemic cell lines, as compared with normal, human primary haemopoietic cells. We show that 30 microM farnesol causes apoptosis of leukaemic cell lines of T- and B-lymphocyte, myeloid or erythroid lineages and primary blasts obtained from patients with AML. However, the same concentration did not kill primary monocytes, or quiescent or proliferating T-lymphocytes. We conclude that farnesol selectively kills AML blasts and leukaemic cell lines in preference to primary haemopoietic cells.