Phi bodies: peroxidatic particles that produce crystalloidal cellular inclusions.

Unique, spindle-shaped particles (phi bodies) and rods with peroxidatic activity are found in certain epithelial cells of normal mice, clofibrate-fed rats, and in leukemic leukocytes. The ellipsoidal shape of phi bodies apparently results from the deformation of spherical granules by extrusion of axial crystalloid that subsequently fragments into rods.

Unusual configurations of endoplasmic reticulum in cells of acute promyelocytic leukemia.

An ultrastructural study of leukemia cells from 8 patients with acute promyelocytic leukemia revealed several features that have not previously been emphasized: prominent dilated rough endoplasmic reticulum and two unusual configurations of endoplasmic reticulum (ER). The two membrane structures, multilaminar ER and complex stellate arrangements of ER, appeared to be morphogenetically related. The multilaminar ER was observed in every mitotic cell and less frequently in interphase cells. The stellate ER complex was observed only in interphase cells. Ultrastructural evidence is presented to support the possible evolution of the stellate ER complex from the multilaminar ER.

Ultrastructural features of basophil and mast cell granulopoiesis in blastic phase Philadelphia chromosome-positive leukemia.

Ultrastructural and ultracytochemical studies were performed on blood and bone marrow specimens from 18 patients with Philadelphia chromosome-positive blastic leukemia; 7 patients were in blast transformation following a typical history of chronic myelogenous leukemia and 11 patients presented with "acute leukemia." The patients were divided into 2 morphologic groups on the basis of light microscopic and cytochemical observations. In group I, which consisted of 11 patients, the proliferating cells were "lymphoid" in appearance and demonstrated many cytochemical, biochemical, and immunologic features similar to those of the lymphoblasts of non-T, non-B acute lymphoblastic leukemia. In group II, which consisted of 7 patients, the proliferating cells were myeloid in appearance. On the basis of ultrastructural observations, the 11 group I patients were divided into 2 subgroups, A and B. Subgroup IA, consisting of 5 patients, was characterized by blasts that demonstrated no differentiating features. In subgroup IB, consisting of 6 patients, 20-30% of the leukemic cells contained inclusions that resembled leukemic mast cell or basophil granules. The leukemic cells in the 7 group II patients manifested myeloid characteristics by light microscopy and prominent basophil and mast cell granulopoiesis by electron microscopy. Abnormalities of other myeloid cell lines were also observed in both the lymphoid and myeloid groups of patients.

Myeloid and megakaryocytic properties of K-562 cell lines.

The expression of myeloid and megakaryocytic markers of differentiation has been studied in one K-562 cell subline, in its clones, and in the original cell line. Cytotoxicity, electron microscopy, immunofluorescence studies with a panel of polyclonal and monoclonal antibodies, and radioimmunoassays were performed on K-562 cells before and after induction with hemin, sodium butyrate, and 12-O-tetradecanoylphorbol-13-acetate. Myeloid membrane markers were present in all K-562 cell lines. Only the early granulopoietic cell surface markers were expressed in 75 to 95% of the cells, while none of the late membrane markers was detected. In contrast, neither the early (myeloperoxidase) nor late (lactoferrin) cytoplasmic markers were present. Thus, K-562 cells showed a membrane phenotype similar to that of a normal or leukemic promyelocyte but lacking myeloperoxidase. Membrane megakaryocytic markers, such as platelet glycoprotein IIIa and platelet peroxidase, were also detected in K-562 cells. However, some other early megakaryocytic markers, such as platelet glycoprotein lb, Factor VIII-R-Ag, and platelet Factor 4, could not be detected by fluorescent labeling. Cloning of the cell line did not result in the selection of a unipotential cell line. These results could be explained by the expression of multilineage markers in a single cell. In all of the cell lines and clones, hemin slightly increased the expression of the myeloid membrane markers without any modification of the megakaryocytic markers. Sodium butyrate and 12-O-tetradecanoylphorbol-13-acetate diminished most of the myeloid markers and very significantly increased the expression of the megakaryocytic markers.

Ultrastructure and cytokinetics of leukemic myeloblasts containing giant granules.

Leukemic myeloblasts containing abnormal granules were studied with ultrastructural, cytochemical, and thymidine labeling techniques to evaluate defects in granulogenesis and proliferation. Giant granules (1 to 3 micron in diameter) and Auer rods were observed in leukemic cells from two patients, and only rarely were both abnormal granule types observed in the same cell. The lysosomal origin of these abnormal granules was demonstrated by their content of peroxidase, esterase, and anionic glycoconjugates. Fusion of small dense granules (less than 0.2 micron in diameter) appeared to be increased in cells containing Auer rods and/or giant granules, but fusion of intact primary granules (0.2 to 0.4 micron in diameter) and sequestration of cytoplasmic contents were observed only in giant granules and not in Auer rods. Although the small granules that fused to form giant granules and Auer rods appeared similar, there was no evidence for transformation of giant granules into Auer rods. In one patient, cells with abnormal granules could easily be distinguished from the larger population of cells that lacked abnormal granules. The perturbation of these two distinct populations by chemotherapy was evaluated with thymidine labeling experiments. A high percentage (2- or 3-fold greater) of the abnormally granulated myeloblasts incorporated tritiated thymidine when compared to myeloblasts without abnormal granules in the same specimen. This difference could have resulted from an underlying metabolic defect which affected both granulogenesis and cell division. These results demonstrate that the formation of giant granules in leukemic cells is morphologically similar to that observed in the Chediak-Higashi syndrome and that leukemic cells with abnormal granules may differ cytokinetically from uninvolved leukemic cells.

Electron microscopic and peroxidase cytochemical analysis of pink pseudo-Chediak-Higashi granules in acute myelogenous leukemia.

Giant round pink inclusions (congruent to 2 micrometers) were seen in neutrophilic myeloblasts, promyelocytes, and myelocytes from three patients with acute myelogenous leukemia. On preliminary examination of the bone marrow smears, these inclusions looked like ingested red blood cells in that they were pink and not azurophilic. The bone marrow specimens were processed for the electron microscopic demonstration of peroxidase with 3,3'-diaminobenzidine and H2O2 at pH 7.6. In all three cases, the inclusions were determined to be large peroxidase-positive granules since they were limited by a single unit membrane and, unlike endocytized red blood cells, were not contained within phagocytic vasuoles. The granules were homogeneously dense for peroxidase and showed no obvious crystalline structure when examined stained or unstained on grid. We believe that they correspond to the giant pink round granules Van Slyck and Rebuck observed in immature leukemic granulocytes in 1974 and termed the pseudo-Chediak-Higashi anomaly. Like the giant purple granules seen in leukemia with this anomaly, these granules also appear to be an abnormal variant of peroxidase-positive azurophil (primary) granules. Their lack of azurophilia is due to the absence of sulfated glycoaminoglycans.

Effects of alkyl-lysophospholipids on human leukemic cell lines measured by nuclear magnetic resonance.

Part of the cytotoxic action of alkyl-lysophospholipids (ALP) on leukemic cells is known to result from the lack of an O-alkyl cleavage enzyme and its antimetabolic effect which results in a toxic lysophospholipid buildup. Further, ALP (5 micrograms/ml) suppresses clonogenicity and tritiated thymidine uptake in HL60 cultures after 24 hr of exposure. The effect of ALP on two leukemic cell lines, HL60 and K562, measured by two nuclear magnetic resonance (NMR) techniques and examined by electron microscopy is reported. 31P-NMR spectroscopy indicates that the adenosine 5'-triphosphate:adenosine 5'-diphosphate ratios are unaffected after 24 hr, as is mitochondrial morphology, judging by electron micrographs. However, cell membrane integrity in HL60 is altered at that time. The earliest ALP effects occur in NMR internal water relaxation at 1 hr after ALP exposure, followed by a small reduction in tritiated thymidine uptake at 4 hr. No effect is observed in K562 cell cultures in morphology or NMR measurements. No new 31P-labeled metabolites were detected in either cell line as a result of drug treatment.

Monoclonal antibody against a unique antigen on human acute promyelocytic leukemia cell line (HL-60).

An IgM kappa monoclonal antibody (WI-1) reacted against HL-60 cells in indirect immunofluorescence and microcytotoxicity tests. It failed to react against 19 other cell lines representing acute myelogenous leukemia, chronic myelogenous leukemia, lymphocytic leukemias, multiple myeloma, Burkitt's lymphoma, monocytoid cells and virus induced lymphoid cell lines. Normal peripheral blood nucleated cells and bone marrow cells derived from acute granulocytic leukemia, chronic granulocytic leukemia, acute lymphocytic leukemia, and chronic lymphocytic leukemia also failed to react with WI-1. Normal peripheral blood lymphocytes, transferred with mitogens, showed no reaction against the antibody. There was some decrease in the reactivity of the cells, against WI-1, following maturation with dimethyl sulfoxide. However, a large percentage of the cells remained positive after maturation. WI-1 reacted specifically against an antigen on the HL-60 cells which had a molecular weight of about 42,000 dalton. Peripheral blood cells from one patient with acute promyelocytic leukemia failed to react with the antibody. It is possible that acute promyelocytic leukemia is an antigenically heterogeneous disease. A large population of acute promyelocytic leukemia patients needs to be tested to see if the specific antigen on HL-60 cells, detected by WI-1, is demonstrable in other patients with acute promyelocytic leukemia.

The acute monocytic leukemias: multidisciplinary studies in 45 patients.

The clinical and laboratory features of 37 patients with variants of acute monocytic leukemia are described. Three of these 37 patients who had extensive extramedullary leukemic tissue infiltration are examples of true histiocytic "lymphomas." Three additional patients with undifferentiated leukemias, one patient with refractory anemia with excess of blasts, one patient with chronic myelomonocytic leukemia, one patient with B-lymphocyte diffuse "histiocytic" lymphoma and one patient with "null" cell, terminal deoxynucleotidyl transferase-positive lymphoblastic lymphoma had bone marrow cells with monocytic features. Another patient had dual populations of lymphoid and monocytoid leukemic cells. The true monocytic leukemias, acute monocytic leukemia (AMOL) and acute myelomonocytic leukemia (AMMOL), are closely related to acute myelocytic leukemia (AML) morphologically and by their response to chemotherapy. like AML, the leukemic cells from the AMMOL and AMOL patients form leukemic clusters in semisolid media. Cytochemical staining of leukemic cells for nonspecific esterases, presence of Fc receptor on the cell surface, phagocytic ability, low TdT activity, presence of surface "ruffles" and "ridges" on scanning EM, elevations of serum lysozyme, and clinical manifestations of leukemic tissue infiltration are features which accompanied monocytic differentiation in these cases.

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.

Variant cell lines from the human promyelocyte line HL60.

The continuous human promyeloid cell line HL60 may be induced to differentiate into neutrophils by the presence of 1.25% dimethylsulphoxide (DMSO) and related compounds [4]. When treated with 12-O-tetradecanoylphorbol-13-acetate (TPA), the cells exhibit may features characteristic of monocytes/macrophages [13]. In both cases, the induced HL60 cells lose their proliferative ability. The availability of this inducible cell line has led to an increasing number of studies aimed at dissecting the process of myeloid differentiation. However, the correct interpretation of such studies may only be reached through the use of appropriate cellular controls; specifically, variant HL60 cell lines which do not differentiate in response to the inducing agents. Here we report the isolation and characterization of two variant cell lines of HL60 which do not differentiate in the presence of 1.25% dimethylsulphoxide but still respond to TPA. Comparison of these HL60 variants with the parental line should facilitate the elucidation of molecular events which regulate myeloid differentiation.

Enhanced polyploidy by glutaraldehyde in cultured HL60 leukemic cells.

Glutaraldehyde 10(-4) M weakened cell proliferation of HL60 cultured cells and enhanced the appearance of giant polyploid cells, up to 32.5% after 6 days. The size and structure of these cells, the quantitative changes in their DNA content with respect to diploid ones demonstrate their polyploid nature, which may be corrected by the occurrence of pluripolar mitoses. However the slowing down of cell proliferation is not enough to orient the cells towards differentiation. Maturation of polyploid cells may be stimulated by retinoic acid and dexamethasone as for diploid ones. Several possible mechanisms of polyploidy are discussed. Except the possibility that the cells may directly fuse, the mechanisms which are considered, may involve a preprophase inhibition, a mitotic arrest at metaphase or a reduction of asters which may result in a defect in cytokinesis, the latter followed by secondary fusion of nuclei.

Acute nonlymphocytic leukemia showing abnormal nuclear lobulation.

An ultrastructural study of four cases of acute nonlymphocytic leukemia with abnormal nuclear lobulation was done. These cases were classified as M3 (Case 1), M3 variant (Cases 2 and 3), and M5, differentiated type (Case 4), on the basis of light microscopy according to the FAB classification. Ultrastructural observation of the leukemic cells of Cases 2 and 3 showed large bundles of fibrils in addition to abnormalities in the endoplasmic reticulum and granules, as had been reported in cases of acute promyelocytic leukemia. Large bundles of fibrils and abnormal nuclear lobulation also seemed to be characteristic of the M3 variant. However, the relationship between these structures was not identified. The exact nature of nuclear lobulation is not known, but seems to be related to some kind of intrinsic force and may be a presentation of the premature occurrence of lobulation intrinsic to the granulocyte or monocyte nucleus.

Microgranular acute promyelocytic leukemia--a case with multiple Auer rods demonstrable only after staining for chloroacetate esterase.

In the past several years there has been increasing recognition of the microgranular variant of acute promyelocytic leukemia (APL). This variant is easily mistaken for other types of acute non-lymphocytic leukemia. Its recognition is important because it carries the same high risk of disseminated intravascular coagulation as typical APL. An important clue to the correct diagnosis is the recognition of small numbers of characteristic cells containing multiple Auer rods. This report presents a case in which such Auer-body-containing cells were demonstrable in the marrow only after staining for chloroacetate esterase. They were not apparent with Wright's stain or Sudan black B. The case also highlights the occurrence of variant APL in children and adolescents.

Expression of terminal deoxynucleotidyl transferase in malignant myeloblasts.

Blast cells from untreated cases of acute leukemia were examined for expression of terminal transferase enzyme (TdT) by immunofluorescence and for myeloid lineage commitment as demonstrated by the presence of myeloperoxidase enzyme at the ultrastructural level. In three cases, an overlapping expression of the "lymphoid-specific" marker TdT was found on peroxidase-positive myeloblasts. These results indicate either the clonal expansion of a rare TdT-positive myeloid precursor or inappropriate expression of TdT by malignant myeloblasts, and further illustrate that TdT expression must be interpreted with caution when distinguishing between lymphoid and myeloid leukemias.

Preleukemic granulocytic sarcomas of the gastrointestinal tract. Report of two cases.

Granulocytic sarcoma, or chloroma, is a tumor composed of immature cells of the myeloid series, which usually occurs as a secondary manifestation of acute myelocytic leukemia. Unique problems in interpretation of these lesions arise when the leukemic picture is absent in peripheral blood and bone marrow. In these cases, granulocytic sarcoma is usually misinterpreted as "reticulum cell sarcoma". Two cases of this neoplasm involving the small intestine and stomach, are reported. Signs of leukemia appeared terminally. The value of cytochemical stains in the differential diagnosis and the possible benefits of early recognition and treatment are emphasized.

Crystal structure of Auer rods in acute myeloblastic leukaemia (AMyL).

Ultrathin sections containing Auer rods from cases of acute myeloblastic leukaemia (AMyL) were tilted in the goniometer stage of the electron microscope and the resulting series of electronmicrographs analysed in an optical diffractometer illuminated by laser. The results showed that Auer rods of AMyL show a truly three dimensional crystal structure. Measurements from the optical diffraction patterns were consistent with a monoclinic unit cell, the unit cell edge lengths a, b, and c being 6.6 [SD) 0.5) nm, 8.6 (0.2) nm, and 9.6 (1.0) nm, respectively; the angle between a and c being 120 (7) degrees. This structure was quite distinct from the "tubular" substructure reported by others in the Auer rods of acute promyelocytic leukaemia (APL), although it was consistent with periodicities measured by others in Auer rods of AMyL. A complete understanding of the three dimensional structures of Auer rods in the different types of acute myeloid leukaemia (AML) could well prove to be of considerable diagnostic importance.

Significance of Phi bodies in acute leukaemia.

Material from 39 patients with acute leukaemia was investigated with the peroxidase cytochemical reaction using 3,3'diaminobenzidine (DAB) and other substrates in order to test their sensitivity in detecting myeloid differentiation. The proportion of positive blasts and of cases with Auer rods in acute myeloid leukaemia (AML) was significantly greater with DAB than with benzidine. In addition, Phi bodies were demonstrated in AML blasts only when DAB was used; Phi bodies were also observed in two out of seven cases of chronic granulocytic leukaemia in "myeloid" blast crisis but were not seen in any case of acute lymphoblastic leukaemia. Phi bodies were more numerous when the reaction was carried out at pH 9.7, and their number was significantly reduced in the presence of 3-amino 1,2,4-triazole. Both findings suggest that the Phi bodies derive from catalase-containing granules (microperoxisomes) and are distinct from Auer rods, which derive from peroxidase-containing (primary) granules. Like Auer rods, Phi bodies appear to be characteristics of immature myeloid cells in leukaemia but are seen with a higher frequency than Auer rods in acute myeloid leukemia.

Acquired lipidosis of marrow macrophages: birefringent blue crystals and Gaucher-like cells, sea-blue histiocytes, and grey-green crystals.

Three varieties of compound lipid inclusions occurring as a secondary phenomenon in marrow macrophages are detectable and distinguishable by Romanowsky staining, ultraviolet fluorescence, and polarised light. Birefringent blue crystals and Gaucher-like cells form one variety, sea-blue granules another, and grey-green crystals a third. All occur chiefly in myeloid leukaemias, either acute or chronic.

Diagnostic and prognostic significance of chromosome abnormalities in marrow and mitogen response of lymphocytes of acute nonlymphocytic leukemia.

Chromosomes of bone marrow from 28 patients with acute nonlymphocytic leukemia (ANLL) (26 with AML, 2 with AMMoL), 19 of whom had chromosome abnormalities, were studied; 11 cases exhibited previously unreported karyotypic abnormalities. The marrows of two cases had 8-21 translocations associated with an iso-X chromosome in the female patient and with 9q13- and a missing Y in the male patient. Usually, AML patients with a 8-21 translocation have been considered to have a good prognosis; however, our cases had rather short survival times. Therefore, the prognosis of AML with an 8-21 translocation but associated with other abnormalities is still not clear. Centromere spreading (CS), which was originally reported in marrow cells of megaloblastic anemia (B12 and folic acid deficiency), was detected in leukemic cells, disappeared during remission, and reappeared on relapse. These findings suggest that CS may be a new type of abnormality in AML. In two patients with atypical hypoplastic anemia and hemolytic anemia, chromosome abnormalities were detected at the anemic stage. One case with CS was associated with atypical hypoplastic anemia and developed AML after 1 year; the other with 48,XY,+i(1q),+3,/12 and -14 had hemolytic anemia and developed AMMoL 3 weeks later. Interestingly, identical clones were detected both before and after the clinical diagnosis of leukemia. These cases strongly support the concept that some chromosome abnormalities precede the clinical manifestations of leukemia. The present study also revealed that lymphocytes in ANLL respond poorly to PHA in the presence of high numbers of blasts but do respond well to mitogens during remission. Therefore, the response of lymphocytes to PHA may serve as one criterion for determining remission.