Ultrastructural analysis of normal and leukaemic cells by the immunogold method and monoclonal antibodies.
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Acute Disease↗
Biomedical subjects
D Catovsky
Publications and source records attributed to D Catovsky.
Reduction in the stearic to oleic acid ratio in leukaemic cells--a possible chemical marker of malignancy.
Total lipid extracts of peripheral blood cells from patients with chronic leukaemias were analysed for relative values of saturation of the eighteen carbon chain length fatty acids (C 18 FA). The results are expressed as saturation index (C 18 S:C 18 U) of the saturated C 18 FA (stearic acid) over the unsaturated C 18 FA (oleic, linoleic and linolenic acids). The saturation indices of the white blood cells (WBC) and the red blood cells (RBC) in specimens from 14 patients with chronic granulocytic leukaemia (CGL) and 17 patients with chronic lymphocytic leukaemias (CLL) were significantly and consistently lower than control specimens. It is proposed that the relative increase in the unsaturated oleic acid could prove to be a chemical marker of malignancy reflecting a deficient cellular control of the process of stearic acid desaturation. The theoretical implications of the implied increase in membrane fluidity for the cells are discussed.
Chromatography, Gas↗
A novel monoclonal antibody BI-3C5 recognises myeloblasts and non-B non-T lymphoblasts in acute leukaemias and CGL blast crises, and reacts with immature cells in normal bone marrow.
A MCA raised against the human acute myelogenous leukaemia cell line KG1 reacted with only KG1 among 26 haematopoietic cell lines covering the major lineages. It reacted with early myeloid (M1/2), 1 of 2 acute myelomonocytic (M4) and most non-B non-T leukaemias, including blast crises of CGL. Among M1-AML cells, both MPO+ and MPO- blasts were BI-3C5+. Blasts in 3 Tdt+ M1-AMLs were simultaneously BI-3C5+. BI-3C5 reacted with 4% cells in normal BM, many of which were histologically recognisable as myeloid precursors. 8-15% of BI-3C5+ cells in BM were simultaneously Tdt+, and all were weakly Ia antigen+. BI-3C5 was unreactive with all peripheral leucocytes, with M3 and M5 AMLs, with lymphoid and myeloid leukaemias of "mature" phenotype (T-ALL, B-ALL, CLL, CGL) and with non-haematopoietic cell lines. BI-3C5 precipitated a 120K moiety from 125I-labelled KG1 membranes. It was not blocked by J5 anti-cALLA. The potential use of BI-3C5 in the classification of acute leukaemias is discussed.
Animals↗
Normal counterparts of hairy cells and B-prolymphocytes in the peripheral blood. An ultrastructural study with monoclonal antibodies and the immunogold method.
The morphological and membrane phenotypic characteristics of normal peripheral blood B-lymphocytes were studied at ultrastructural level by means of the immunogold method with the following McAb: anti-HLA-Dr (OKIa and FMC4), FMC7, OKT10, and alpha HC1 and alpha HC2, reactive with HCL cells. Five morphological subsets of B-lymphocytes were identified: (i) 40% had a high nuclear cytoplasmic ratio, well condensed chromatin and small pale granules localised in one area of the cytoplasm, these cells expressed HLA-Dr and did not react with any of the other McAb; (ii) 40% had a prominent nucleolus, variable amount of chromatin condensation and localised granules, these cells were HLA-Dr positive and some of them expressed FMC7 as do the cells of B-PLL; (iii) 10% had a villous outline and variable numbers of polyribosomes scattered throughout the cytoplasm, some of these cells resembled morphologically HCL cells and were HLA-Dr +, FMC7 +, alpha HC1 +, alpha HC2 + and OKT10 +/-. The remaining two types of B-cells (10%) had the morphology of antibody secreting cells with parallel arrays of endoplasmic reticulum and some displayed electron dense granules in the Golgi zone. These cells were: (iv) lymphoplasmacytoid, the more common type, which were HLA-Dr +/-, FMC7-, alpha HC1-, alpha HC2 + and OKT10 +/-, and (v) plasma cells which were positive only with OKT10. The similarities in morphology and membrane markers between two of the subsets defined in this study and the cells of B-PLL and HCL suggest that they may represent normal counterparts of these B-cell malignancies.
Adult↗
Monocytic differentiation induced by 1,25 dihydroxyvitamin D3 in myeloid cells: an ultrastructural immunocytochemical study.
We have studied, by ultrastructural morphology and immunocytochemistry, the alterations that occur in cells from the HL60 leukaemia cell line and from patients with CGL following incubation in vitro with 1,25(OH)2D3 for 2-5 days. The main morphological changes observed were in the nuclear shape, the development of autophagic vacuoles and the appearance of a population of small granules in the cytoplasm. These changes were associated with a significant reduction in MPO activity and increased expression of membrane antigens detected by the monocyte-specific McAb FMC17 and FMC32, as shown by the IGM at EM level, and a decrease in granulocyte-specific antigens demonstrated by the McAb FMC10. These observations suggest that promyelocytes and myelocytes could transform into monocyte-like cells and that this remodelling of cells was associated with autophagic digestion of cellular structures.
Antibodies, Monoclonal↗
Functional and phenotypic analysis of a T cell prolymphocytic leukemia.
Peripheral blood mononuclear cells obtained from a patient with prolymphocytic leukemia expressed the surface membrane markers characteristic of resting mature T helper lymphocytes. These cells responded to the T cell mitogens PHA and Con A in a blast transformation assay but not the anti-T cell monoclonal antibody Leu 4 and the B cell mitogen, PWM. The concentration of PHA or Con A eliciting maximum blast transformation was less than that required by normal mononuclear cells. The leukemic cells recognised and responded to allogeneic pooled mononuclear cells in a mixed lymphocyte culture. In addition, although they did not express Ia antigens, they served as effective stimulators in the mixed lymphocyte reaction. Consistent with the helper phenotype, the leukemic cells did not produce suppressor factors, but provided help for normal B-enriched lymphocytes to respond to PWM as assessed by both blast transformation and IgG production. T lymphocyte colonies developed when the leukemic cells were treated with PHA during a 20 h liquid culture prior to being seeded into semisolid agar medium containing either PHA or an IL2-containing lymphokine. There was no growth when untreated cells were seeded directly into IL2-containing agar. Analysis of colony formation indicated that, as with normal resting T lymphocytes, proliferation occurred in two distinct steps; activation in response to PHA and replication in response to IL2-like growth factors. These findings demonstrate that in this case the helper T prolymphocytes have the functional capabilities of normal mature T lymphocytes as predicted from their helper phenotype.
Aged↗
Atypical promyelocytic leukemia (M3) with immature primary granules and t(15;17).
An unusual case of acute myeloid leukemia with a standard t(15;17) is described. While light microscopy morphology was suggestive of acute myeloid leukemia M5a and light microscopy cytochemistry showed 80% of blasts to be strongly positive with Sudan Black B--more consistent with a diagnosis of M4--ultrastructural analysis demonstrated that the predominant cells were promyelocytes with immature primary granules hardly visible with the Romanovsky stains by light microscopy. Because typical cytologic and clinical features of M3 or M3 variant were lacking this atypical case would not have been recognized but for the presence of t(15;17) and the demonstration of promyelocytic features by electron microscopy.
Chromosome Banding↗
Ceftazidime as first-line therapy for fever in acute leukaemia.
Fifty patients with acute non-lymphocytic leukaemia were treated by random allocation with either ceftazidime alone or a combination of piperacillin, netilmicin and cefotaxime for 65 febrile neutropenic episodes. Nineteen of 33 patient episodes (58%) responded to ceftazidime alone compared with 21 of 32 episodes (66%) treated with the combination. There was one infective death in a patient given the combination; rates of documented superinfection were low. The treatment groups appeared identical in terms of patient demography, underlying disease and other risk factors, though patients with a clinical site of infection responded more slowly than those without. Bacteraemia per se did not appear to influence outcome. Bactericidal serum concentrations greater than or equal to 8 X the minimum bactericidal concentration were predictive of a rapid response (within 4 days) to antibiotics. Furthermore, serum from patients treated with ceftazidime maintained adequate cidal activity against Pseudomonas aeruginosa for longer than that obtained from patients treated with the three-drug combination. Ceftazidime was shown to be a safe and effective alternative to the three-drug combination for the initial management of febrile neutropenic episodes in leukaemic patients.
Adolescent↗
Blast crisis of chronic granulocytic leukemia with mast cell and basophilic precursors.
The authors report a patient with Ph1-positive chronic granulocytic leukemia (CGL) who developed "blast crisis" after six years of chronic phase. The presence of mast cell precursors and basophil blasts was demonstrated by ultrastructural morphology and cytochemistry. Membrane phenotype studies with monoclonal antibodies helped in the further characterization of these cells. The possible implication of these findings in the origin of mast cells and the relationship of these cells with basophils are discussed.
Adult↗
Monoclonal antibody OKT17 recognizes most cases of T-cell malignancy.
Membrane phenotype analysis with monoclonal antibodies (McAb) has demonstrated great heterogeneity within the T-cell malignancies. We describe here the reactivity with an anti-T cell McAb, OKT17, in 80 leukaemia samples. Cells from all types of T-cell leukaemia (48 cases), except from the small group of pre-T-ALL, strongly expressed the antigen identified by OKT17 whereas none of the 32 non-T leukaemias were OKT17 positive. When the reactivity of OKT17 was compared with that of other pan-T markers, OKT17 was positive in a larger number of T-cell leukaemias: 87% of cases compared with 74% with E-rosettes and 73% with the McAb 3A1. In the mature or post-thymic proliferations OKT17 was positive in 96% of cases, compared with 77% with E-rosettes and 61% with 3A1. The latter reagent, on the other hand, was better than OKT17 for detecting leukaemias with a thymic phenotype, 100% and 68% of positive cases respectively. The combined use of OKT17, 3A1 and terminal transferase permits a more precise classification of all the T-cell leukaemias according to the main stages of T-cell differentiation.
Antibodies, Monoclonal↗
Characterization of blast cells in chronic granulocytic leukaemia in transformation, acute myelofibrosis and undifferentiated leukaemia. I. Ultrastructural morphology and cytochemistry.
A systematic analysis of the blast cell population was carried out on samples from 50 patients suffering from blast transformation of chronic granulocytic leukaemia (CGL) (31) and of myelofibrosis (4), acute myelofibrosis (AM) (11) and undifferentiated acute leukaemia (4). Transmission electron microscopy (TEM), used in 41 samples, included: morphology and techniques for myeloperoxidase (MPO), platelet-peroxidase (PPO) and acid phosphatase (AP). The majority of cases were also studied by light microscopy cytochemistry and with a battery of cell markers which are reported in the accompanying paper (San Miguel et al, 1985). The characterization of the type(s) of proliferating blasts was made from the integration of ultrastructural and immunological data. TEM morphology allowed the precise recognition of specific granules in basophil and mast-cell precursors and of ferritin particles in blasts of erythroid lineage; these rare cell types were not adequately characterized by other methods. The PPO reaction made possible the identification of pure megakaryoblastic proliferations in 38% of cases, including eight of the 11 with AM; megakaryoblasts were also present in nine of 12 cases with mixed blast cell types. The MPO and AP reactions were useful for the characterization of myeloblasts and monoblasts, respectively. Lymphoblasts could be distinguished from other cell types by TEM morphology and negative MPO and PPO reactions. TEM techniques were valuable for diagnosing correctly the type of blast cell in this study in which only four cases (8%) remained unclassifiable.
Acute Disease↗
Interferon is effective in hairy-cell leukaemia.
Seventeen patients with hairy-cell leukaemia (HCL) and peripheral cytopenias were given human lymphoblastoid interferon (Wellferon), 3 megaunits daily or 6 megaunits on alternate days intramuscularly, for 4-24 weeks. Twelve of the patients had undergone splenectomy, three had no palpable spleen and had therefore not been offered surgery, and two patients with substantial splenomegaly were given interferon (IFN) as treatment of first choice. Toxic effects were minor except in one patient who experienced a severe form of somnolence syndrome. In all patients hairy cells (HCs) were cleared from the blood and platelet and Hb levels improved in 2-14 weeks. Neutrophils were improved in 14/17 of the patients. In the two patients with splenomegaly, the spleen became impalpable after 5-8 weeks therapy, and haematological improvement occurred at 12-14 weeks. HC infiltration of the marrow was reduced in all patients, but was complete (less than 5%) in only two, both of whom had impalpable spleens. Immunological surface-marker studies confirmed that light-chain-restricted B cells disappeared from the blood in parallel with the clearance of morphological HCs. There was no evidence of HC maturation and no increase in phenotypic NK cells. T cells were moderately reduced and the relatively greater reduction of Leu 2a+ suppressor cells resulted in increased Leu 3a+/2a+ helper/suppressor ratios in 11/17 of the patients. Early experience in the six patients who have stopped IFN suggests that, after an initial further increase in Hb and neutrophil levels, HCs gradually return with slow deterioration of haematological parameters. Interferon is now the treatment of choice for patients becoming cytopenic post-splenectomy or for patients without splenomegaly. IFN is effective first-line therapy in patients with splenomegaly, but further work is needed to establish whether the agent should replace splenectomy in such patients. Some form of maintenance or re-treatment therapy will probably be necessary.
Adult↗
Recommended methods for cytological procedures in haematology. International Committee for Standardization in Haematology (ICSH).
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3,3'-Diaminobenzidine↗
Hypereosinophilic syndrome preceding T cell lymphoblastic lymphoma.
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Adult↗
T lymphoblastic transformation of refractory anaemia with excess of blasts.
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Aged↗
Osteolytic lesion as the presenting feature of chronic granulocytic leukaemia.
We describe a woman in whom the first manifestation of chronic granulocytic leukaemia (CGL) was an osteolytic lesion. Six months later the peripheral blood showed the picture of CGL in blast crisis. Chromosome studies confirmed the diagnosis and immunological and ultrastructural studies demonstrated the presence of two blast populations: basophil blasts and megakaryoblasts.
Aged↗
Dipeptidylaminopeptidase IV (DAP IV) in B- and T-cell leukaemias.
A cytochemical study in samples from 100 lymphoid leukaemias, 84 of B-cell type and 16 of T-cell type, was carried out with three acid hydrolases: DAP IV, acid phosphatase (AP) and alpha-naphthyl acetate esterase (ANAE). DAP IV was studied in leukaemic T-cells both by cytochemistry and by a monoclonal antibody with the immunoperoxidase technique. Both methods showed similar results. AP and ANAE gave weak reactions in immature B-cell leukaemias (common-ALL and B-CLL) and were strongly expressed in plasma cell disorders. DAP IV showed no activity in any of the types of B-cell leukaemia studied and was strongly positive in some T-cell leukaemias but with a more restricted distribution than ANAE and AP. T-lymphoblasts (T-ALL) and mature (T8+) leukaemias were DAP IV negative. Within the T4+ malignancies DAP IV was positive in four T-prolymphocytic leukaemias, one of two T-CLL and one of three Sezary syndrome cases. Although DAP IV is strictly T-cell specific it does not appear to aid the differentiation between B- and T-cell disorders or the identification of T-cell subsets as determined by monoclonal antibodies. It remains to be established whether this enzyme will define a functionally distinct T-cell subset.
Acid Phosphatase↗
Bone marrow biopsy changes in acute myeloid leukaemia. I: Observations before chemotherapy.
Pretreatment bone marrow trephine biopsy sections (BMB) from 34 patients with acute myeloid leukaemia (AML) were studied in parallel with bone marrow aspiration smears and peripheral blood films. In four cases marrow aspiration was inadequate and in five cases it was unsatisfactory. In two other cases hypoplastic AML was diagnosed, the aspirate in one suggested hypercellularity and in another it was unsatisfactory. Trephine biopsy was superior to aspiration for the evaluation of fat and marrow cellularity, pattern and extent of blast cell infiltration, homogeneity of the leukaemic infiltrate, frequency of mitoses, residual haemopoietic activity and presence of inflammatory cells. Of the various features studied in the sections, the presence of an increased number of plasma cells and considerable myelodysplasia (MD) appeared to be unfavourable prognostic features. We conclude that trephine biopsies are essential for the diagnosis of hypoplastic AML and are most useful when marrow aspiration is either inadequate or unsatisfactory. They also provide additional information about the bone marrow changes in AML and suggest that some histological features may also have prognostic significance.
Biopsy↗