Ultrastructure and Cytokinetics of Leukemic Myeloblasts Containing Giant Granules1

[CANCER RESEARCH 39. 3834-3844. October 1979] 0008-54 727 79/0039-OOOOS02.00 Ultrastructure and Cytokinetics of Leukemic Myeloblasts Containing Giant Granules1

Richard T. Parmley,2 Gary V. Dahl, Ronald L. Austin, Patsy A. Gauthier, and Francis R. Denys

Institute of Dental Research. Departments ot Pathology and Pediatrics, University of Alabama in Birmingham, Birmingham, Alabama 35294 Â¡R.T. P., R. L. A., F. R DI, and Division of Hematology/Oncology, St Jude Children s Research Hospital. Memphis, Tennessee 38100Â¡G. V. D.. P. A. G.]

ABSTRACT with Auer rods. Since "pseudo-Chediak-Higashi" granules in leukemic leukocytes have not been studied ultrastructurally, a Leukemic myeloblasts containing abnormal granules were detailed comparison with abnormal granulogenesis in the Che studied with ultrastructural, cytochemical, and thymidine label diak-Higashi syndrome has not been possible. ing techniques to evaluate defects in granulogenesis and pro Although the Chediak-Higashi-like granules are an acquired liferation. Giant granules (1 to 3 /im in diameter) and Auer rods manifestation of the leukemia, patients with the Chediak-Hi were observed in leukemic cells from two patients, and only gashi syndrome may develop lymphoma or myeloproliferative rarely were both abnormal granule types observed in the same and accelerated lymphoma-like disorders (9, 33), which sug cell. The lysosomal origin of these abnormal granules was gest the presence of an underlying defect that affects both demonstrated by their content of peroxidase, esterase, and granulogenesis and cell division. The increased destruction anionic glycoconjugates. Fusion of small dense granules (less of leukocytes with abnormal granules in the Chediak-Higashi than 0.2 jum in diameter) appeared to be increased in cells syndrome may result in increased proliferation (8); however, containing Auer rods and/or giant granules, but fusion of intact [3H]dThd3 labeling studies have not been evaluated in this primary granules (0.2 to 0.4 Â¡imin diameter) and sequestration disorder, nor have previous studies demonstrated proliferative of cytoplasmic contents were observed only in giant granules differences in leukemic cells with abnormal granules. and not in Auer rods. Although the small granules that fused to The present study of the morphological characteristics and form giant granules and Auer rods appeared similar, there was the proliferative activity in relationship to these characteristics no evidence for transformation of giant granules into Auer rods. was undertaken to identify defects in granulogenesis and pro In one patient, cells with abnormal granules could easily be liferation and to determine if these abnormalities were related distinguished from the larger population of cells that lacked to those described for the Chediak-Higashi syndrome. abnormal granules. The perturbation of these two distinct pop ulations by chemotherapy was evaluated with thymidine label MATERIALS AND METHODS ing experiments. A high percentage (2- or 3-fold greater) of the abnormally granulated myeloblasts incorporated tritiated Patients. Patient 1 was a 9-year, 10-month-old white girl thymidine when compared to myeloblasts without abnormal who was well until 2 weeks prior to the diagnosis of acute granules in the same specimen. This difference could have myelocytic leukemia, when she developed symptoms of bruis resulted from an underlying metabolic defect which affected ing and leg pain. Patient 2 was a 13-month-old white girl both granulogenesis and cell division. These results demon diagnosed to have acute promyelocytic leukemia after a 4- strate that the formation of giant granules in leukemic cells is week history of increasing petechiae and anemia. After diag morphologically similar to that observed in the Chediak-Higashi nosis, both patients underwent 4 to 8 weeks of induction syndrome and that leukemic cells with abnormal granules may therapy, which included vincristine, actinomycin D, 1-/?-D-ara- differ cytokinetically from uninvolved leukemic cells. binofuranosylcytosine, 6-azauridine, u-asparaginase, and methotrexate. Despite this intensive therapy, bone marrow INTRODUCTION aspirates from the patients continued to demonstrate replace ment of the marrow space with leukemic cells. Patient 1 de Abnormal granulogenesis is frequently observed in myeloid veloped severe coagulation problems and died of an intracra- cells from patients with leukemia and myeloproliferative disor nial hemorrhage 5 weeks after diagnosis. Patient 2 expired 21 ders (4, 32, 41 ). Although Auer rods were initially described as weeks after diagnosis with progressive disease complicated by abnormal granules in lymphoid leukemia (3), they have sub severe liver dysfunction and hemorrhage. sequently been recognized as abnormal primary granules Light Microscopy. In addition to routine Wright's staining of which form exclusively in acute nonlymphocytic leukemia (1). blood and marrow smears, the following cytochemical proce Ultrastructural studies of Auer rods have disclosed a unique dures were performed: (a) ethanol-formalin-fixed smears were periodicity of dense material, the presence of acid phosphatase stained for the demonstration of peroxidase according to the (19, 20, 38), and an abnormal packing and concentration of method of Kaplow (25); (fa) naphthol AS-D chloroacetate ester peroxidase (4, 6, 11). Giant granules resembling those de ase was demonstrated in buffered formalin-fixed smears, using scribed in the Chediak-Higashi syndrome (12, 22) have also a naphthol AS-D chloroacetate solution (Sigma Chemical Co., been observed in leukemia and myeloproliferative disorders St. Louis, Mo.) as described by Yam et al. (40); (e) a-naphthyl (18, 26, 34), but they have not been observed in association acetate esterase was demonstrated in buffered formalin-fixed ' Supported by NIH Grants DE-02670, CA-15956. and CA-020180 and by smears, using an a-naphthyl acetate solution (Sigma) as de- ALSAC. â€¢'Towhom requests for reprints should be addressed

Received March 5, 1979; accepted June 19. 1979. 3 The abbreviation used is: [3H]dThd. tritiated thymidine.

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Downloaded from cancerres.aacrjournals.org on September 30, 2021. © 1979 American Association for Cancer Research. Giant Granules in Leukemic Cells scribed by Yam et al. (40); (d) aldehyde fuchsin (basic fuchsin, of blasts, and both giant granules and Auer rods were observed Lot 752459; Fisher Scientific Co., Pittsburg, Pa.) staining of in 0.3% of cells. Abnormal granules were easily identified in acid glycoconjugates was accomplished on formalin-fixed Wright's stained preparations as reddish purple, needle-like or smears as described previously (1 7). round giant granules but were more frequently observed in Thymidine Labeling. After written consent, bone marrow aldehyde fuchsin-stained specimens (Fig. 1). For optimal stain samples from both patients were studied on 6 occasions during ing of acid mucosubstance, the aldehyde fuchsin solution was the first week of therapy as part of an ongoing protocol to allowed to ripen overnight at room temperature, was filtered, evaluate the effects of chemotherapy on leukemic cell kinetics. and was used within 48 hr. Giant granules also demonstrated Bone marrow from the posterior iliac crest was aspirated into strong reactivity for peroxidase, naphthol AS-D chloroacetate heparinized syringes, and 1 ml of the aspirate was incubated esterase, and Sudan black. In Patient 1, 80% of blasts dem at 37Â°with 1 /iCi of [3H]dThd (1.9 Ci/mmol; Schwarz/Mann, onstrated naphthol AS-D chloroacetate esterase staining, and Orangeburg, N. Y.) for 1 hr with intermittent agitation. The cells 12% of blasts contained a-naphthyl esterase staining. In Patient were rinsed, routinely smeared on glass coverslips, and air 2, 90% of blasts stained for naphthol AS-D chloroacetate dried. The specimens were dipped in NTB2 emulsion and held esterase, and 10% stained for a-naphthyl esterase. Leukemic for 14 days in the dark. The slides were then developed with blasts lacked alkaline phosphatase or periodic acid-Schiff reac Kodak D-19 and Kodak rapid-fix solutions. Cells containing 5 tivity. or more labels were considered to be labeled. The percentage of labeled blasts was then determined by counting 1000 cells Thymidine Labeling in a Wright's stain preparation, and the percentage of labeled The labeling index (determined by counting 1000 Wright's cells containing giant granules was determined in an aldehyde stained blasts) in Patient 1 varied from 7 to 11 % during the fuchsin stain preparation (17). first week of chemotherapy, whereas in Patient 2 the values Electron Microscopy. A portion of the heparinized marrow varied from 3 to 7%. Background labeling was minimal, and no aspirate was transferred to 1-ml glass tubes and centrifuged at labeling was observed in specimens not exposed to [3H]dThd. 1000 x g for 3 min to separate the buffy coat. The buffy coats were fixed and minced in 3% glutaraldehyde, pH 7.35-0.1 M As reported previously by this laboratory (31 ), a single labeling cacodylate buffer for 1 hr at 4Â°.This was followed by 3 rinses index has an inherent experimental error, which was estimated by a separate analysis of variance of 50 triplicate labeling index in 0.1 M cacodylate buffer (7 g/dl sucrose), pH 7.35. determinations for marrow leukemia blasts. Using this method, In addition to morphological preparations, a portion of the specimen was processed for demonstration of intrinsic perox- a difference in the labeling index is significant at the 0.05 level if 2 successive labeling indices exceed 1.86 or are less than idase according to the method of Graham and Karnovsky (21 ). The substrate medium consisted of 10 ml of 0.05 M Tris-HCI 0.54 of the other determination. buffer, pH 7.6, saturated with 3 mg of 3,3'-diaminobenzidine In Patient 1, aldehyde fuchsin staining allowed differentiation of cells containing normal and abnormal granules, and labeling with 0.01% H2O2 (3 drops of 3% H2O2 added immediately indices for each of these populations were determined (Fig. 2; before use). A portion of each specimen was incubated for 30 Chart 1). Only 1 to 3% of the blasts in each sample contained min at room temperature in the complete substrate medium. To abnormal granules. The labeling index of cells with abnormal achieve better stain penetration, some specimens were first incubated overnight at 4Â°in medium lacking H2O2, followed by granules was determined by counting an average of 170 cells per timed sample (1020 cells for the entire 168-hr period). In a 30-min incubation in the complete medium. Control speci mens were processed similarly except that H2O2 or the diaminobenzidine was omitted.

The morphological and cytochemical preparations were All ILASTl D rinsed in buffer and postfixed for 1 hr in 1% OsCv The NORMALGRANULI specimens were then routinely dehydrated with alcohol and AINOIMAI GRANULE propylene oxide and embedded in Spurr low-viscosity medium. Thin sections of morphological preparations were counterstained with uranyl acetate and lead citrate, whereas thin sections of cytochemical preparations were not counterstained. The sections were viewed with a Philips 300 electron microscope at an accelerating voltage of 60 kV.

RESULTS

Light Microscopy

In Patient 1, giant granules (1 to 4 per cell; up to 3 firn in Ã• 3 diameter) were observed in 2.5% of bone marrow blasts, whereas Auer rods (1 or 2 per cell) were present in 0.2% of blasts. The majority of blasts from Patient 1 did not contain HOURS FROM START OF THERAPY (ciuci ADMINIITIIID) abundant cytoplasmic granules. In contrast, most of the blasts Chart 1. Labeling indices for all leukemic cells, leukemic cells with giant from Patient 2 contained numerous cytoplasmic granules, and granules, and leukemic cells with normal granules. The labeling index of blasts with abnormal granules was significantly higher in all except the 168-hr specimen. 30% of the blasts contained one to several (as many as 15) VCR DAUNO. vincristine-daunomycin; ARA-C. 1-/J-D-arabinofuranosylcytosine; Auer rods. Giant granules (1 to 4 per cell) were present in 4% 6 AZUR. 6-azauridine.

OCTOBER 1979 3835

Patient 2, the heavy granulation and intense staining of the cent endoplasmic reticulum and other cytoplasmic organelles blasts prevented accurate differentiation of these subpopula- (Fig. 10), similar to those previously described in some cases tions. The labeling index of cells with abnormal granules was of myelocytic leukemia (28). Some segments of endoplasmic consistently greater than the labeling index of cells with normal reticulum appeared to be excessively dilated and contained granules. It was significantly elevated in all except the 168-hr membrane material. sample. Cytochemistry. The diaminobenzidine reaction product was often observed in primary granules, small dense granules, Electron Microscopy megagranules (Figs. 11 and 12), Auer rods (Figs. 13 and 14), and membrane-limited circular profiles (Fig. 15). Although stain penetration into tissue blocks was improved in specimens Morphology. Leukemic blasts in both patients contained exposed to the diaminobenzidine overnight (Figs. 13 to 15), nuclei with dispersed chromatin and large nucleoli. Although the majority of leukemic cells resembled myeloblasts or pro- similar staining of individual cell organelles was observed with the 30-min exposure to the staining solution (Figs. 11 and 12). myelocytes with round nuclei, abundant endoplasmic reticu- Infrequent staining was observed in Golgi lamellae and endo lum, and distinct primary granules (Fig. 3), several cells evi plasmic reticulum. Small dense peroxidase-positive granules denced monocytic characteristics such as irregularly folded (less than 0.2 /im in diameter) were frequently observed con nuclei and small pleomorphic granules. Cytoplasmic maturation often exceeded nuclear maturation and resulted in nuclear- tacting and/or fusing with Auer rods (Figs. 13 and 14) and cytoplasmic asynchrony as described previously (5-7). megagranules (Fig. 11). Larger peroxidase-positive primary granules were occasionally observed contacting megagranules Primary granules 0.2 to 0.4 jum in diameter were observed in but not Auer rods. Some Auer rods and megagranules lacked various maturational stages in the majority of cells (Fig. 3). the diaminobenzidine reaction product despite the presence of Immature granules contained a moderately dense core with an electron-lucent rim, whereas mature granules were uniformly reaction product in nearby normal and abnormal granules of the same cell. dense. In addition, small dense granules, less than 0.2 /im in No reaction product was evident in normal and abnormal diameter, were frequently observed in these cells and appeared granules or circular profiles of cells exposed for 30 min to increased when compared to descriptions of normal myeloid control solutions lacking the H2O2 or diaminobenzidine (Fig. cells (5, 36). No secondary granules could be identified in 16). Specimens exposed to the diaminobenzidine overnight leukemic blasts. without subsequent addition of H2C>2contained cells with a Several cell profiles contained one or 2 megagranules which moderate amount of dense reaction product in the cytoplasmic were membrane limited and were up to 3 /im in diameter. Some granules. This density was not evident in overnight controls in of these granules contained a uniformly dense nucleoid (Fig. 3), and some contained less dense material, whereas others which the diaminobenzidine was omitted from the solution. contained membrane-like material or entrapped cytoplasmic organelles (Fig. 4). DISCUSSION Abnormal maturation of primary granules was observed in some leukemic cells. These cells had segmented mature nuclei These studies have compared the formation of Auer rods with primary granules that often contained myelin figures or and giant granules in leukemic myeloblasts and identified as other heterogeneous material (Fig. 5). sociated abnormalities in granulogenesis. Both abnormal gran The morphological appearance of Auer rods was more uni ules appear to evolve from exaggeration of the normal process form than that observed for giant granules. These granules (5, 36) of coalescence and fusion of small primary granule were membrane limited and were up to 4 /Â¿mlong and 1 /tm precursors and Golgi-derived vesicles. Although abnormal fu wide (Fig. 6). The granules usually contained electron-dense sion of granules has been observed in leukemic cells containing material in the periphery and lucent or extracted material in the Auer rods (6, 11, 20, 32) and in leukocytes with giant granules center. Many Auer rods contained a crystalline structure with from patients with the Chediak-Higashi syndrome (15, 37, 39), periodic dense material similar to that described previously the observation in this study of both types of abnormal granules (11). in the same specimen has provided a unique opportunity for Cells observed at the ultrastructural level rarely contained investigation of abnormal granulogenesis. We have observed both Auer rods and giant granules (Fig. 7). Cells with a similar that granule fusion resulting in Auer rod formation appears finding were also infrequently seen with the light microscope. limited to the small dense granules less than 0.2 /im in diameter, Abnormal granules were not observed in eosinophils, baso- whereas giant granule formation encompasses this process as phils, platelets, or megakaryocytes. well as fusion of intact primary granules and frequent entrap Golgi vesicles and small dense granules often contacted ment of cytoplasmic material. Thus, the latter process may each other, as well as giant granules (Fig. 7), Auer rods, and result from more extensive cellular dysfunction. normal-appearing primary granules. Entrapment of cytoplasmic Unlike Auer rods, the formation of giant granules or pseudo- material and contact and/or fusion with intact primary granules Chediak-Higashi granules occurs only rarely in leukemic mye (0.2 to 0.4 /urn in diameter) were observed only in association loblasts. To our knowledge, only 2 cases have been reported with megagranules (Fig. 7) and not with Auer rods. Fusion of in the literature (34), and, although the ultrastructural features small dense granules resulted occasionally in the formation of of these granules were not investigated, the ultrastructure of a membrane-limited circular (possibly spherical) profile which giant platelet granules observed in some cases of myeloid enclosed other cytoplasmic organelles (Figs. 6 and 8). leukemia has been related to the Chediak-Higashi anomaly Segments of endoplasmic reticulum often contacted adja (26). The granules observed here in leukemic blasts were

3836 CANCER RESEARCH VOL. 39

Downloaded from cancerres.aacrjournals.org on September 30, 2021. © 1979 American Association for Cancer Research. Giant Granules in Leukemic Cells histochemically and ultrastructurally similar to the abnormal granule enlargement, similar or possibly identical to that of primary granules of patients with the Chediak-Higashi syn giant granule precursors, does occur prior to condensation of drome. The presence of a limiting membrane and their content the granule contents to form the Auer rod. The lack of morpho of peroxidase and esterase clearly distinguish the giant gran logical or quantitative evidence for a transition from giant ules in this study from Dohle bodies (41) or other giant inclu granules to Auer rods in this study suggests that, although sions previously reported in a myeloproliferative disorder (13). these granules share some abnormalities in granulogenesis, The morphology of the leukemic cell giant granules is similar they mature to form 2 distinct abnormal granule types. Alter to giant granules previously demonstrated in the Chediak-Hi natively, a transition of Auer rods to giant granules could occur gashi syndrome (15, 37, 39), in which large granules are with progressive cellular dysfunction and autophagy of Auer synthesized as a single intact primary granule or form as a rods. However, the rare occurrence of pseudo-Chediak-Hi- result of continued fusion of primary granules and/or small gashi granules in myelocytic leukemia, despite the relatively dense granules (15) and entrapment of cytoplasmic contents frequent observation of Auer rods, would suggest that, if this (37, 39). Although proposed explanations for the abnormal process occurs, it is very infrequent. granule fusion in the Chediak-Higashi syndrome include altered The presence of cytokinetic differences in these cells is intracellular cyclic nucleotides and dysfunction of microtubules indicated by their difference in nucleoside incorporation, which (8, 27) and/or granule membranes (39), additional studies are persisted even under the influence of chemotherapy. The required to define underlying defects leading to the abnormal marked difference between the [3H]dThd labeling of leukemic granule fusion in leukemic and Chediak-Higashi leukocytes. blasts with and without giant granules in Patient 1 may indicate The nature of the increased small dense granules in these that a relatively larger proportion of the cells with abnormal specimens is not known. A similar increase in small granules granules was undergoing DMA synthesis. The high labeling has been observed in neutrophils of mink with Chediak-Higashi index could result from rapid cell division with few resting cells disease (15). The reactivity of these small granules with dia- or from a slowing of the DMA synthesis time with accumulation minobenzidine, pH 7.4, suggests a Golgi-derived origin similar of cells in the S phase of the cell cycle. Alternatively, there may to that of peroxidase-containing primary granules rather than be accelerated production of giant granules during the later an origin similar to that of catalase-containing peroxisomes that part of interphase which is subsequently related to death of demonstrate similar staining at higher pH levels (24). these cells as they pass into the early d phase. This would The distribution of the diaminobenzidine reaction product in result in a decrease in G, and G0 cells and a relative increase the abnormal granules is similar to previous observations lo in S-phase cells. Accelerated death of myeloid cells in Chediak- calizing peroxidase in Auer rods of leukemic cells (1, 4, 11) Higashi disease (8) is consistent with this hypothesis. and giant granules in Chediak-Higashi leukocytes (22). The Recent studies of Chediak-Higashi leukocytes (10, 27) have diaminobenzidine reaction product presumably localizes per shown both excessive amounts of cyclic adenosine 3':5'-mono- oxidase in neutrophil primary granules (16); however, this phosphate, which is known to stimulate the flow of cells into S reagent also reacts with hemoglobin and other heme proteins phase (2, 35), and simultaneous dysfunction of microtubules under similar conditions (21). The interpretation of the reaction (23, 27, 29), which may prevent cells from completing a normal is more specific for enzyme activity after a 30-min incubation mitosis and further accentuate an accumulation in late inter- with a negative control solution lacking H2O2, as done for some phase. Similar abnormalities may exist in leukemic cells with specimens in this study. Although the overnight incubation with giant granules and result in perturbations of both granuloge the diaminobenzidine improves stain penetration in the speci nesis and the cell cycle. The additional observation of malig men, the "control" specimen lacking H2O2 also demonstrates nant degeneration of leukocytes in the Chediak-Higashi syn some staining. This may result from localization of a broader drome (9, 33) further suggests an underlying metabolic defect range of heme proteins or may indicate the presence of some which could alter normal cell growth and division. Conceivably, H2O2 intrinsic to the specimen. The lack of peroxidase staining the presence of giant granules in leukemic cells could herald in a few Auer rods and giant granules with both incubation the emergence of a second rapidly dividing cell clone; however, times is consistent with the peroxidase deficiency reported for the consistently low percentage of affected cells despite pro abnormal primary granules in some cases of myelocytic leu gression of the disease would argue against this possibility. kemia (14). Alternatively, the finding could result from masking Nevertheless, the findings in this study of defective growth and of the peroxidase by another component of the granule or division evidenced by giant granule formation and increased extraction of the enzyme during tissue processing. The possi [3H]dThd labeling in the same cell population suggest a unique bility that some of the peroxidase-negative giant granules orig deviation of these cells from normal control mechanisms. inate from secondary granules similar to those recently ob served in the Chediak-Higashi syndrome (30) would seem REFERENCES unlikely in these patients, since secondary granulogenesis was not observed in the leukemic blasts. 1. Ackerman. G. A. Microscopic and histochemical studies on the Auer bodies The findings of numerous giant granules and few Auer rods in leukemic cells. Blood, 5. 847-863, 1950. 2. Armato, U., Draghi, E., and Andreis, P. G. Effects of purine cyclic nucleotides in one patient, numerous Auer rods and few giant granules in on the growth of neonatal rat hepatocytes in primary tissue culture. Exp. another patient, and the infrequent occurrence of both granule Cell Res., 105: 337-347, 1977. 3. Auer, J. 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OCTOBER 1979 3837

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E.. and Grauer, M. J. Giant eosinophilic granules in the chronic Di 37. White. J. G. The Chediak-Higashi syndrome: cytoplasmic sequestration in Guglielmo syndrome. N Engl. J. Med., 274. 209-210, 1966. circulating leukocytes. Blood, 29. 435-451. 1967. 19. Freeman, J. A. The ultrastructure and genesis of Auer bodies. Blood, )5: 38. White, J. G. Fine structural demonstration of acid phosphatase activity in 449-465. 1960. Auer bodies. Blood. 29 (Suppl.). 667-682. 1967. 20. Freeman, J. A. Origin of Auer bodies. Blood, 27. 499-510, 1966. 39. White, J. G. The Chediak-Higashi syndrome: fine structure of giant inclusions 21. Graham, R. C.. Jr.. and Karnovsky, M. J. The early stages of absorption of in freeze-fractured neutrophils. Am. J. Pathol.. 72: 503-519. 1973. injected horseradish peroxidase in the proximal tubules of mouse kidney: 40. Yam, L. T., Li, C. Y., and Crosby, W. H. Cytochemical identification of ultrastructural cytochemistry by a new technique. J. Histochem. Cytochem., monocytes and granulocytes. Am. J. Clin. Pathcl., 55. 283-290, 1971. 14: 291-302, 1966. 41. Zucker-Franklin, D. Physiological and pathological variations in the ultra- 22. Higashi, O. Congenital gigantism of peroxidase granules. Tohoku J. Exp. structure of neutrophils and monocytes. Clin. Haematol,, 4 485-508, 1975.

The specimens for Figs. 2,3,8. 11. 12, 15. and 16 were obtained from Patient 1, and those for Figs. 1. 4 to 7, 9, 10. 13, and 14 were obtained from Patient 2. Figs. 1 and 2 are light micrographs, and Figs. 3 to 16 electron micrographs. The thin sections for Figs. 1 to 10 were counterstained with uranyl acetate and lead citrate, whereas those for Figs. 11 to 16 were not stained with heavy metals. Specimens for Figs. 11 and 12 were incubated for 30 mm in the complete diaminobenzidine substrate medium, whereas specimens for Figs. 13 to 15 were first incubated overnight in the diaminobenzidine solution without H2O2, followed by exposure to complete substrate medium. Fig. 16 is of a control specimen incubated for 30 min in the diaminobenzidine solution lacking H2O2. Fig. 1 Aldehyde fuchsin strongly stains Auer rods (left) and giant granules (.right). Rarely, both abnormal granule types were observed in the same cell, x 2.300. Fig. 2. Silver grains, indicative of [JH)dThd incorporation, are evident over the nucleus of this leukemic cell containing an aldehyde fuchsin-reactive giant granule (arrow) x 2.300. Fig. 3. This leukemic myeloid cell contains a large giant granule (thick arrow) with a central dense nucleoid 3 /im in diameter. There are several dilated segments of rough endoplasmic reticulum (ER) and a prominent Golgi complex (G). A moderately enlarged vacuole containing variably dense material (thin arrow) is present near the Golgi face opposite the giant granule and may represent a precursor form of the giant granule. Several primary granules appear to be maturing normally; immature granules (Pi) contain a variably dense center with an electron-lucent periphery that decreases in moderately mature granules (P2) and is not seen in fully mature granules (P3) in which dense material extends to the limiting membrane, x 17.000.

3838 CANCER RESEARCH VOL. 39

x?Â» I

OCTOBER 1979 3839

Downloaded from cancerres.aacrjournals.org on September 30, 2021. © 1979 American Association for Cancer Research. Fig. 4. A giant inclusion in this cell contains heterogeneous membrane-limited material, x 40.000. Fig 5 Rare leukemic cells, such as this, demonstrate apparent condensation of nuclear chromatin and segmentation of nuclear lobes. Granule maturation is. however, grossly abnormal in that most primary granules appear degenerated and contain myelin figures (arrows), x 19,000. Fig. 6. This leukemic cell contains several enlarged and elongated granules that correspond to Auer rods sectioned longitudinally (L), perpendicularly (P). or tangentially (T). The central portions of the granules appear less dense and may have been extracted during tissue processing, x 20.000 Inset, an almost circular (possibly spherical) profile that appears to result from fusion of small dense granules, x 51.000

3840

Fig. 7. This leukemic cell contains a number of abnormal granules, some of which resemble Auer rods and one of which contains heterogeneous material similar to that observed in some giant granules (inset). A few granules (arrows) are tangentially sectioned and may represent branched or Y-shaped variants of the Auer rod. x 26.000; inset, 65,000.

OCTOBER 1979 3841

â€” â€¢, . ' â€¢â€¢â€¢HFig. 8. Golgi lamellae (G) adjacent to a giant granule containing a central nucleolo1 (fn/ck arrow) and heterogeneous material. The inclusion is contacted by a primary granule (P) below and several Golgi vesicles (thin arrows') above Adequate fixation of this cell is evidenced by the preservation of the nuclear envelope and mitochondria (M), x 40.000. Fig. 9. Concentric (possibly spherical) profiles of membrane-bound dense material are present in this leukemic cell. Additional circular profiles are in the periphery of the cell and are associated with coalescence of small dense granules (arrows), x 42,400 Fig. 10. The circular profiles of rough endoplasmic reticulum (arrows) closely approximate each other, forming a lamellated structure x 28.100.

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Fig. 11. Dense diaminobenzidine reaction product stains primary granules as well as a giant granule (arrow). The nucleoid of the megagranule contains fine dense reaction product and is surrounded by a more heterogeneous flocculent reaction product, x 22,600 Fig. 12. A diaminobenzidine-reactive megagranule (arrow) is present in this mitotic leukemic cell with an anaphase distribution of chromosomes (C). x 17.000. Fig. 13. Several diaminobenzidine-reactive small granules appear to be fusing with this Auer rod. x 4200.

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f ER \

Fig. 14. Periodic arrays of dense diaminobenzidine reaction product are present in perpendicular (P) and longitudinal (L) sections of Auer rods in this leukemic cell, x 28,100. Inset, a small granule fusing with one of these structures, x 70,000 Fig. 15. Dense diaminobenzidine reaction product is present in these circular profiles (cl. Fig. 9), suggesting a relationship to aberrant primary granulogenesis. One structure encloses a mitochondrion (arrow). Dilated segments of endoplasmic reticulum (ER) lack enzymatic reaction product, x 69,300. Fig. 16. In this control specimen incubated for 30 min in a diaminobenzidine solution without added H2O2. megagranules (arrows) lack the density observed in cytochemical preparations (cf Figs. 11 to 15). x 20.500.

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Richard T. Parmley, Gary V. Dahl, Ronald L. Austin, et al.

Cancer Res 1979;39:3834-3844.

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