Electron Microscopic and Peroxidase Cytochemical Analysis of Pink Pseudo-Chediak-Higashi Granules in Acute Myelogenous Leukemia1
Total Page：16
File Type：pdf, Size：1020Kb
[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 ¡R.J. K.J; and Department of Pathology. University of California School of Medicine. San Francisco. California 94143 ¡D.F. B.¡ 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 DECEMBER 1980 4473 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 4°, 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, pred- nisone, 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.