Electron Microscopic and Peroxidase Cytochemical Analysis of Pink Pseudo-Chediak-Higashi Granules in Acute Myelogenous Leukemia1

Home , Auer rod, Inclusion bodies

[CANCER RESEARCH 40, 4473-4481, December 1980] 0008-5472/80/0040-4473S02.00 Electron Microscopic and Peroxidase Cytochemical Analysis of Pink Pseudo-Chediak-Higashi Granules in Acute Myelogenous Leukemia1

William A. Dittman, Robert J. Kramer, and Dorothy F. Bainton2

Sacred Heart Medical Center, Spokane. Washington 99024 [W. A. D.]; Station 5. Pasco. Washington 99301 Â¡R.J. K.J; and Department of Pathology. University of California School of Medicine. San Francisco. California 94143 Â¡D.F. B.Â¡

ABSTRACT We have had the opportunity to study 3 patients with AML whose blasts, promyelocytes, and myelocytes contained enor Giant round pink inclusions (=2 jam)were seen in neutrophilic mous round pink inclusions, initially thought to be ingested myeloblasts, promyelocytes, and myelocytes from three pa RBC. Analysis by electron microscopy and peroxidase cyto tients with acute myelogenous leukemia. On preliminary ex chemistry revealed that these pink structures were large per amination of the bone marrow smears, these inclusions looked oxidase-positive granules and therefore also represented ab like ingested red blood cells in that they were pink and not normal variants of the azurophilic (primary) granule population. azurophilic. The bone marrow specimens were processed for In addition, these 3 cases did not have DIC. the electron microscopic demonstration of peroxidase with 3,3'-diaminobenzidine and H2C>2at pH 7.6. In all three cases, the inclusions were determined to be large peroxidase-positive MATERIALS AND METHODS granules since they were limited by a single unit membrane Case Studies and, unlike endocytized red blood cells, were not contained within phagocytic vacuoles. The granules were homogeneously Case 1. A 62-year-old white housewife with a history of dense for peroxidase and showed no obvious crystalline struc hypertension and arthritis was sent for a hematological con ture when examined stained or unstained on grid. We believe sultation in June 1974 because of fatigue, anemia, and recent that they correspond to the giant pink round granules Van onset of bruising. Physical examination revealed pallor and Slyck and Rebuck observed in immature leukemic granulocytes multiple ecchymoses but not organomegaly or adenopathy. in 1974 and termed the pseudo-Chediak-Higashi anomaly. Like Laboratory examination of the blood revealed pancytopenia the giant purple granules seen in leukemia with this anomaly, (Table 1), but no abnormal circulating leukocytes were de these granules also appear to be an abnormal variant of per tected. A bone marrow aspiration and biopsy specimen were oxidase-positive azurophil (primary) granules. Their lack of hypercellular, showing an increase in the granulocytic series, azurophilia is due to the absence of sulfated glycoaminogly- 5% myeloblasts, and promyelocytes containing Auer rods and cans. salmon-pink inclusion bodies. The pink inclusions were usually large (1 to 2 firn) and round, but they were occasionally tear INTRODUCTION shaped or tapered at both ends. Rarely, both forms were present in a single cell. The patient's condition was diagnosed In 1974, Van Slyck and Rebuck (40) observed, on Wright's- as AML. stained smears, giant round granules in leukemic cells from 2 Over the next 2.5 years, the patient was given various patients with acute myelomonocytic leukemia. Since this ac treatments for AML and had a smoldering course without quired morphological abnormality closely mimicked the giant complete remission. The Auer rods disappeared with partial round granules seen in the Chediak-Higashi anomaly, a rare remission, but the giant inclusions merely decreased in num autosomal recessive disorder, they termed it the pseudo-Che ber. Initially, the patient was treated with daunomycin, vincris- diak-Higashi anomaly of acute leukemia. Since some of the tine, prednisone, and 1-/?-D-arabinofuranosylcytosine. Be giant granules stained azurophilic while others were pink, Van cause she came near death twice and her marrow remained Slyck and Rebuck hypothesized that they represented abnor abnormal, she was switched to a maintenance dose of daily malities of azurophilic (primary) and specific (secondary) gran cyclophosphamide and monthly vincristine. During the ensuing ule populations, respectively. More cases of this anomaly as months, she had a number of complications, mainly infections. sociated with leukemia have now been reported (16, 18, 20, A bone marrow specimen was processed for fine-structural 26, 36, 38), and three cases with purple-staining giant granules examination in November 1975. have been thoroughly studied by electron microscopy and In October 1976, the patient was found to have an endo- peroxidase cytochemistry (30, 39). These investigators dem metrial adenocarcinoma, which was treated with radiation ther onstrated that these dark giant granules were indeed abnormal apy. At that time, her WBC count was 0.7 x 109/liter. She variants of the peroxidase-positive azurophilic granule popu developed bowel obstruction and died on January 8, 1977. An lation. Interestingly, their clinical courses were complicated by autopsy revealed metastatic carcinoma to the peritoneum, liver, DIG.3 pleura, and bone. The marrow was hypercellular. During the ' This investigation was supported by Grant CA-14264 from the National course of her illness, there was no clinical or laboratory evi Cancer Institute, Department of Health. Education and Welfare dence of DIC. 2 To whom requests for reprints should be addressed. Case 2. A white 16-year-old female was found to have AML 3 The abbreviations used are: DIG, disseminated intravascular coagulation; in May 1976. She had experienced dizziness on exertion, easy AML, acute myelogenous leukemia; RER. rough endoplasmic reticulum. Received May 13. 1980; accepted September 12. 1980. bruising, and heavy menstrual flow for 6 weeks. Her WBC

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Downloaded from cancerres.aacrjournals.org on September 26, 2021. © 1980 American Association for Cancer Research. W. A. Dittman et al. count was 5.0 x 109/liter with a marked shift to the left in the (19, 30) and dialyzed iron (17, 35). Cells from bone marrow neutrophilic series, including 20% myeloblasts. Her blood data aspirates were fixed in 1.5% glutaraldehyde in 0.1 M sodium are summarized in Table 1. Her bone marrow was hypercellular cacodylate buffer (pH 7.4) with 1% sucrose for 10 min at 4Â°, and showed granulocytic hyperplasia with a marked shift to the washed twice in the same buffer with 7% sucrose, and shipped left. The myeloblasts contained large salmon-pink cytoplasmic on ice by air freight from Washington to California. They were inclusions and occasional typical azurophilic Auer rods. Culture incubated for peroxidase with 3,3'-diaminobenzidine and H2O2 for Philadelphia chromosome was negative. Leukocyte alkaline at pH 7.6 by the method of Graham and Karnovsky (21), phosphatase was normal. Bone marrow was obtained for elec postfixed in 1% OsO4, and subsequently processed as previ tron microscopic examination in May 1976. ously described (7). The patient was treated with thioguanine, vincristine, prednisone, 1-yS-D-arabinofuranosylcytosine, and cyclophospha- RESULTS mide and was in remission for 6 months. During this period, no giant granules were seen in her granulocytic precursor cells. A The question of whether the large round pink inclusions seen bone marrow specimen taken in January 1977 showed 14% on smears in early neutrophilic precursor leukocytes (Fig. 1, myeloblasts but no giant granules. The patient did not respond inset) were exogenous particles (i.e., ingested RBC) or endog to reinduction therapy and died in April 1977 of a pulmonary enous products of the leukocytes was easily resolved by elec infection. There was no evidence of DIG. tron microscopy. These peroxidase-positive inclusions (Fig. 1) Case 3. In January 1976, the preoperative leukocyte differ were each surrounded by a single unit membrane (Fig. 2). If ential for a white 52-year-old male scheduled to undergo they were indeed ingested RBC, there would be 2 membranes, elective surgery revealed abnormal circulating leukocytes with an inner one belonging to the RBC and an outer one belonging large round pink inclusions. About 12% of the cells were blast- to the phagocytic vacuole. like and contained very large round pink granules. The physical The cells with the large pink inclusions which were up to 3 examination revealed no abnormalities. The complete blood mmin diameter also contained normal peroxidase-positive azur- count is recorded in Table 1. An aspirate of sternal bone ophil (primary) granules (Fig. 3a), which in humans are usually marrow showed granulocytic hyperplasia and the same large no larger than 500 nm. Some cells contained many giant pink granules in blasts and promyelocytes. Some blasts con granules (Fig. 1), whereas others contained only one (Fig. 3a). tained typical azurophilic Auer rods. A bone marrow specimen Although the content of these granules was not completely was obtained for electron microscopy in March 1976. By May homogeneous, since there were both light and dark areas, we 1976, the patient had become symptomatic with fatigue and were unable to resolve crystalline structures even on unstained bleeding and had a falling hematocrit and an increase in his material. This lack of crystalline structure differs from the case circulating blasts to 50%. He received chemotherapy for the previously reported by Tulliez ef al. (39). When examined by first time on March 9, 1976. Initially, he was given cyclophos- light microscopy, the giant granules showed no staining for phamide, thioguanine, prednisone, and vincristine every 4 aldehyde fuchsin (Fig. 3a, inset), nor dialyzed iron (not illus weeks, but, beginning in November 1977, he received the trated), whereas the adjacent normal azurophil granules were treatment regimen every 6 weeks. His last hematology values, stained. recorded on August 29, 1980, show a WBC count of 6.3 x Having demonstrated that the giant inclusions were abnormal 109/liter, including 1% bands, 51% polymorphonuclear leu peroxidase-positive granules of endogenous origin, we wanted kocytes, 32% lymphocytes, and 12% monocytes. His hema to further delineate their formation. In previous studies of tocrit was 45%, and his platelet count was 140 x 109/liter. A normal human bone marrow (5, 7), we had demonstrated that recent marrow specimen still showed large pink inclusion bod the peroxidase-positive azurophilic, or primary, granules are ies in many cells of the granulocytic series. There has been no formed by the usual pathway followed by secretory products evidence of DIG during the course of his illness, and he is still (29) (RER â€”Â»vesicles-Â»Golgi complex â€”Â»storagegranules); alive as of October 1980. i.e., in normal promyelocytes, the marker enzyme peroxidase is synthesized and segregated within the RER and transported Light and Electron Microscopy to the Golgi region by small transitional vesicles. The enzyme is concentrated within the Golgi cisternae (see Ref. 4, diagram); Smears of bone marrow were stained for aldehyde fuchsin then small Golgi-derived vesicles bud off and move to the

Table 1 Summary of initial blood data for 3 patients with AML seriesPatient123Age621652SexFFMHemato-crit Neutrophilic

+ poly- mor- phonu- clearneutro

philicleuko (xlOVliter)3721Leukocytecount(xlOVliter)1.15.05.7Myeloblasts(%)2012Promyelocytes(%)151Myelo-cytes(%)101Meta-myelo-cytes(%)51Bands Eosino-(%) (%)251644Plateletcount cytes10252Lymphocytes(%)904127Mono-cytes(%)73 phils

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Downloaded from cancerres.aacrjournals.org on September 26, 2021. © 1980 American Association for Cancer Research. Abnormal Granules in AML peripheral cytoplasm where they aggregate to form the larger One major difference between our 3 cases and others was mature azurophil granules. In the 3 bone marrow specimens the color of the giant granules as observed by light microscopy. described here, PER and Golgi cisternae contained no reaction In the earlier reports, the granules were described as purple product for peroxidase (Fig. 3a). This lack of reaction product (39), reddish purple (30), or azurophilic (18), whereas the may well reflect the procedures used, rather than a true ab inclusions that we observed were pink. Furthermore, these sence of peroxidase, since 3 days elapsed between fixation of inclusions did not stain for aldehyde-fuchsin, a stain for anionic the specimens in Washington and incubation for the enzyme in glycoconjugate, or dialyzed iron, a stain for carboxylated and California. However, it is interesting to note that other investi sulfated acidic glycoconjugates. In contrast, the giant reddish- gators have had difficulty demonstrating peroxidase in PER purple inclusions described by Parmley et al. (30) stained and Golgi cisternae in leukemic cells containing giant granules strongly for aldehyde-fuchsin. Since Dunn and Spicer (1 7) and (30, 39). Clustered near the giant granules in our specimens Parmley ef al. (31) have demonstrated that the azurophilia of were large numbers of small vesicles containing high concen normal azurophil (primary) granules is due to the presence of trations of peroxidase (Fig. 3b). Parmley ef al. (30) have sulfated glycoaminoglycans, we hypothesize that the giant suggested that the giant granules in leukemic cells are formed nonazurophilic granules we observed lack acid mucosub- by the fusion of these small dense vesicles as well as by the stance(s). It should be mentioned that these giant round gran fusion of intact primary granules and concomitant entrapment ules in acute leukemia stain for other constituents of normal of cytoplasmic material. The 2 abnormal granule forms in Fig. azurophil granules, such as peroxidases (18, 26, 30, 39), 2 (labeled ag) may exemplify the latter mechanism. naphthol AS-D chloroacetate esterase (26, 30), acid phospha- In addition to the prominent round inclusions, the cells from tase (18, 26), Sudan black (30), and periodic acid-Schiff (16, 2 patients contained several other forms of abnormally large 18, 26). peroxidase-positive granules (Fig. 4). These granules were Schmalzl ef al. (36) have noted that Auer bodies also usually usually elongated, and some contained amorphous densities stain for peroxidase, naphthol AS-D chloroacetate esterase, (Fig. 4a). Others contained a crystalloid structure (Fig. 4b), acid phosphatase, and Sudan black and are thus closely re and yet others contained an obvious well-organized crystal lated to normal azurophil granules. They point out, however, (Fig. 4c). The structure containing the crystal could be seen by that not all Auer bodies in a particular specimen exhibit all of light microscopy on Wright's-stained smears and was azuro- these staining properties. This variability of staining and pre philic with a rounded middle and pointed ends (Fig. 4c, inset). sumably of granule content seems to apply to the pseudo- All these forms seem to be variants of Auer bodies. The form Chediak-Higashi anomaly as well. illustrated in Fig. 4c was recently termed Phi body by Hanker The 2 investigations of giant granules in leukemic cells by et al. (22, 23). Since Auer originally described and illustrated electron microscopy and peroxidase cytochemistry (30, 39) organelles of these shapes, we prefer to call them Auer bodies have both shown that: (a) the granules are bound by a single (see Ref. 3, Fig. 15). unit of membrane, and the vast majority contain a matrix The following observations in more mature neutrophils were densely reactive for peroxidase; (b) swarms of small peroxi made. Although the giant granules were observed only in dase-positive vesicles are prominent in the cytoplasm of cells myeloblasts, promyelocytes, and myelocytes, other abnormal containing such granules; and (c) peroxidase reaction product ities were seen in mature neutrophils. The most striking of the is not clearly demonstrable in RER or Golgi cisternae in these pathological abnormalities was the paucity and sometimes cells. There are, however, some major discrepancies among complete absence of peroxidase-negative specific granules the findings. In the study by Tulliez ef al. (39), no Auer bodies (Fig. 5). This secondary granule population is usually formed were observed. The giant granules contained crystalline cores, during the myelocyte stage and ordinarily constitutes two-thirds seen best on unstained grids, and exhibited a periodicity dif of the total granule population of human neutrophils (7). In ferent from that of the typical Auer bodies seen in acute addition, there were prominent nuclear blebs, which have been promyelocytic leukemia associated with DIG. The giant gran reported to be more common in leukemic cells (2), usually ules frequently contained heterogeneous membranous material blasts, than in normal cells. and amorphous nucleoids. In our specimens, the granule matrix was relatively homogeneous and did not have a crystalline DISCUSSION substructure. Furthermore, our patients showed no evidence of DIG. Like Parmley ef al. (30), we observed Auer rods, but Abnormal forms of granules have been observed in the early only rarely were both abnormal granule types seen in the same myeloid cells of patients with acute leukemia by many investi cell. gators (1, 3, 5, 6, 8, 9, 11 -14, 24, 25, 36, 37, 41 ). The first None of the investigations thus far, including our own, has form recognized and the one best characterized to date is the provided clear evidence of the mechanism by which the giant Auer rod (3), which is present in the myeloblasts and promye granules are formed. Tulliez ef al. (39) have suggested that locytes of from 10 to 15% of patients with AML. This cytoplas they are formed by fusion of azurophil granules. Parmley ef al. mic inclusion is usually needle shaped, although Auer also (30) have hypothesized that they are formed by 2 processes: commented on other forms, and is easily recognized on smears (a) the fusion of small peroxidase-positive vesicles; and (b) the prepared with Romanowsky-type stains because it is azuro- fusion of intact primary granules and concomitant entrapment philic (i.e., stains reddish purple); it also stains for peroxidase of cytoplasmic material. It should be mentioned that these and several other enzymes (6, 8, 12, 14, 36). Only 7 cases of numerous small peroxidase-positive vesicles (Fig. 3b) were "giant lysosome-like structures" (26), "oval cytosomes" (36), also a prominent feature in cells forming Auer rods (Fig. 4) as or pseudo-Chediak-Higashi granules (18, 30, 39) in cells from previously observed (6, 12). Also, perhaps, the previously patients with acute leukemia have been previously studied. mentioned absence of stainable glycoaminoglycans in these

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large granules may provide a clue to their formation. Even sence of specific (secondary) granules in neutrophils from though the factors responsible for the concentration of en patients with AML (4, 6), and Odeburg ef al. (27) and Rausch zymes and their packaging into normal azurophil granules are et al. (33) have observed the absence or marked decrease of still obscure, we believe that they may be similar to the factors lactoferrin, a substance found exclusively in the specific gran suggested by Reggio and Palade (34) to explain the concen ule population. These 2 characteristics may permit recognition tration of enzymes in zymogen granules of the pancreatic of mature neutrophils derived from leukemic clones. We have exocrine cell. These investigators proposed that the concen observed these abnormal neutrophils quite frequently in acute tration was not achieved by membrane ion pumps in the con myeloid leukemia with maturation, i.e., the M2 variety, as well densing vacuoles but possibly occurred as a result of the as in certain cases of hematopoietic dysplasia. We plan to osmotic effects of a sulfated polyanion, a peptidoglycan. Hy- follow patients with the latter condition to see whether the pothetically, this polyanion together with secretory proteins presence of such neutrophils correlates with the eventual ap that are predominantly cationic could cause a reduction in the pearance of acute leukemia. osmotic activity within the condensing vacuoles; the resultant Several of the patients in previously published case reports efflux of water would bring about concentration. Additional had acute promyelocytic leukemia with clinical and laboratory data on normal human leukocytes are needed before abnor findings suggestive of DIG (30, 36, 39). In this study, we have malities in the condensation of azurophil granules in leukemic described 3 more patients, all with well-differentiated AML, cells can be further analyzed. whose neutrophilic promyelocytes and myelocytes contained The formation of the megagranules in the hereditary Chediak- giant pink granules. Unlike the patients previously studied, they Higashi anomaly has been carefully studied by Davis et al. (15). showed no definite evidence of DIG. Promyelocytes from persons and animals with this trait lack We have no explanation at present for these different clinical normal azurophil (primary) granules and, instead, contain en findings. In addition, the survival of more than 3 years in Case larged pleomorphic granules, which may resemble azurophil 3 is atypical of AML and deserves further comment. Despite granules to varying degrees. These unusual granules are not persistent bone marrow morphology consistent with the diag present in the blasts and become apparent in promyelocytes nosis of AML and the presence of megagranules in many after the appearance of the precursor vesicles derived from neutrophilic precursor cells, the peripheral blood remains rel Golgi cisternae. The precursor vesicles undergo a series of atively normal. This case may therefore be similar to the one fusions leading to the formation of the megagranules. In hu previously described by Branda et al. (11) in which there was mans, the characteristic crystalloid does not seem to form, and an indolent course as well as a marked dichotomy between the the granule matrix develops a reticulated array of membrane- blastic morphology of the bone marrow and a relatively normal like material. These granules also contain peroxidase and stain blood picture. In addition, the blasts of their patient contained with Sudan black. On Wright's staining of smears, they may prominent vacuoles and frequent Auer rods. Furthermore, stain reddish purple, blue, purple, or sometimes pink (10, 15, when the bone marrow was cultured, the leukemic cells differ 32, 39, 40). The second stage of granulopoiesis, which gives entiated to form abnormal primary granules which appeared to rise to the peroxidase-negative specific (secondary) granule, rupture and cause cytolysis of these cells. On the basis of such is unaffected; and a full complement of these granules is observations, they suggested that the indolent clinical course present in mature neutrophils. No normal azurophil granules might be related to blast destruction within the marrow and are present. It was recently demonstrated that some form of recommended less intensive chemotherapy. It is reasonable to the megagranules also contains lactoferrin, indicating some postulate that our Case 3 has intramedullary destruction of limited fusion with specific granules (32). The defects under leukemic cells. lying the abnormal granule fusion in Chediak-Higashi leuko cytes have not yet been defined although 2 possibilities, alter REFERENCES ation of intracellular cyclic nucleotides and dysfunction of microtubules and granule membranes, have been investigated 1. Ackerman. G. A. Morphology and cytochemistry of the granule-vacuole body of leukemic cells. Blood. 12: 443-447. 1957. (see Ref. 28). It should also be remembered that, in hereditary 2. Ahearn. M J., Trujillo. J. M.. Cork. A.. Fowler. A., and Hart. J. S. The Chediak-Higashi syndrome, other hematopoietic cell types association of nuclear blebs with aneuploidy in human acute leukemia. contain megagranules as do many other nonhematopoietic Cancer Res., 34: 2887-2896. 1974. 3. Auer. J. 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Fig. 1. Electron micrograph of a neutrophilic promyelocyte from bone marrow, stained for peroxidase. The cytoplasm contains at least 6 abnormally large round granules (arrows), the pseudo-Chediak-Higashi granules, in addition to normal-size azurophil granules (ag). Very small peroxidase-positive vesicles (ve) are present near some of the large granules, nu. nucleolus: m, mitochondria: er, endoplasmic reticulum. x 17.500. Inset, light micrograph of a Wright-Giemsa-stained bone marrow preparation showing 2 large pink inclusions in a promyelocyte. N, nucleus, x 3,000. Fig. 2. Higher magnification of part of a neutrophilic promyelocyte from bone marrow, stained for peroxidase. Pseudo-Chediak-Higashi granule (p-CH) is limited by a single unit membrane (me), similar in thickness to the membranes of the adjacent azurophil granules (ag). Although the matrix of the pseudo-Chediak-Higashi granule is not entirely uniform, no crystalline structure could be detected even on unstained specimens, er, endoplasmic reticulum. x 67.000. Fig. 3. Electron micrographs of neutrophilic promyelocytes from bone marrow, stained for peroxidase. In a, note the abnormally large (about 1 firn) round peroxidase-positive granule (p-CH) in addition to many smailer normal-size (about 500 nm) azurophil granules (ag). The perinuclear cisterna (pn), endoplasmic reticulum (er), and Golgi cisternae (Gc) are not reactive for peroxidase. x 16.000. Inset, light micrograph of an aldehyde fuchsin-stained preparation showing normal reactive granules (a-0 and 2 large unstained megagranules (arrows), x 1.300. In b, the cluster of small reactive vesicles (ve) can be seen to better advantage. These vesicles probably fuse (ve') to form the larger granules (p-CH). Other azurophil granules (ag) are of normal size, m, mitochondria; N. nucleus, x 40.000. Fig. 4. Portions of the cytoplasm of myeloblasts and promyelocytes, illustrating several other abnormal forms of peroxidase-positive granules. The granules in a and o may represent immature forms present before full crystallization of granule contents has occurred, as in the elongated form in c. Numerous small vesicles (ve) are present near these forming granules, ag. normal azurophil granules; m. mitochondria; er, endoplasmic reticulum; pn, perinuclear cisterna, a, x 22.000; b. x 39.000; c, x 27.000. Inset, light micrograph showing an elongated granule (arrow) like the one in c. Such granules were azurophiHc. x 1,600. Fig. 5. Electron micrograph of a more mature neutrophil from bone marrow, stained for peroxidase. Although the nuclear chromatin is condensed and segmented (s) and the cell is presumably a mature polymorphonuclear neutrophilic leukocyte, its cytoplasm contains very few peroxidase-negative specific granules (sq). In a normal mature polymorphonuclear neutrophilic leukocyte, about two-thirds of the granules would be peroxidase-negative specific granules, and about one-third would be peroxidase-positive azurophils (ag). Some of these peroxidase-positive granules are also abnormal in that they tend to be larger and less homogeneous in content than normal azurophilic granules. A nuclear bleb (D) is also present. A/I and N?. nuclear segments; m, mitochondria, x 20.000.

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Downloaded from cancerres.aacrjournals.org on September 26, 2021. © 1980 American Association for Cancer Research. Electron Microscopic and Peroxidase Cytochemical Analysis of Pink Pseudo-Chediak-Higashi Granules in Acute Myelogenous Leukemia

William A. Dittman, Robert J. Kramer and Dorothy F. Bainton

Cancer Res 1980;40:4473-4481.

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