Megakaryocyte Abnormalities in Thrombotic Thrombocytopenic Purpura LAWRENCE KASS, M.D. Department of Internal Medicine, Simpson Memorial Institute, The University of Michigan, Ann Arbor, Michigan 48105 ABSTRACT Kass, Lawrence: Megakaryocyte abnormalities in thrombotic thrombocytopenic purpura. Am. J. Clin. Pathol. 61: 639-644, 1974. Morphologic and enzymatic abnormalities were found in megakaryocytes obtained from bone marrow of four patients with thrombotic thrombocytopenic purpura. These abnormalities included large megakaryocytes resembling those in essential thrombocythemia, abnormalities in nuclear segmentation and "doughnut-hole" nuclei, multiple cytoplasmic vacuoles, erythrophagocytosis by megakaryocytes, small "reticuloid" megakaryocytes, and markedly increased activity of cytoplasmic LDH. These observations suggest the possibility of an intrinsic and perhaps neoplastic abnormality of megakaryocytes in thrombotic thrombocytopenic purpura. (Key words: Megakaryocyte; Purpura, thrombotic thrombocytopenic; Erythrophagocytosis; Lactic dehydrogenase.) THIS REPORT describes abnormalities of megakaryocytes in the bone marrow of four patients with thrombotic thrombocytopenic purpura (TTP). Materials and Methods Sternal or iliac bone marrow was obtained from four patients with the classic "triad" of thrombotic thrombocytopenic purpura comprised of hemolytic anemia, thrombocytopenia, and neurologic findings. All of these patients succumbed to the disorder, and at necropsy were found to have the typical capillary hyaline thrombotic lesions of TTP in various organs. Received September 17, 1973; received revised manuscript November 21, 1973; accepted for publication December 6, 1973. Supported by the Elizabeth Roodvoets Memorial Grant for Cancer Research of the American Cancer Society (CI-79), and by a grant from the National Cancer Institute (USPH CA 14428-01). Films of bone marrow flecks were made on methanol-cleaned glass coverslips and stained with Wright's stain for light microscopic examination. Other, unstained, coverslips were stained for lactic dehydrogenase (LDH) activity according to the method of Stuart and Skowron 10 and counterstained with nuclear fast red. 5 Control coverslips for LDH activity included marrows from three presumably normal individuals, three patients with agnogenic myeloid metaplasia, ten patients with idiopathic thrombocytopenic purpura (ITP), and four patients with essential thrombocythemia. Results In all of the TTP specimens, megakaryocytes were increased in number. Although some of the megakaryocytes appeared indistinguishable from normal 639
640 KASS A,J.C.P. Vol. 61 J FIG. 1. Unusually large megakaryocyte with multilobulated nucleus and abundant richly granular cytoplasm containing several sequestrations of granular material. Wright's stain, x 1,200. megakaryocytes, the majority of them appeared abnormal. The abnormalities were found in all the marrows examined, and seemed to be classifiable into several types. First, unusually large megakaryocytes were observed (Fig. 1). These megakaryocytes frequently contained multilobulated nuclei and voluminous, richly granular cytoplasm. Occasionally, the cytoplasm contained large areas of sequestered granules, as seen in Figure 1. A second type of abnormal megakaryocyte is illustrated in Figure 2, A and B. This megakaryocyte appeared normal in size, but the configuration of the nucleus was abnormal. Partially-disconnected nuclear lobes were seen, and numerous dense aggregates of chromatin appeared in the nucleus. This type of megakaryocyte frequently had a doughnut-shaped nucleus, with a small, central "hole," as illustrated in Figure 2, A and B. A third type of abnormal megakaryocyte is seen in Figure 2,C and D. This megakaryocyte had an eccentrically-located, coarsely-reticular appearing nucleus and its cytoplasm was filled with numerous vacuoles. Few granules were observed, and little evidence of platelet formation at the periphery of the megakaryocyte cytoplasm could be found. A fourth type of abnormal megakaryocyte appears in Figure 3, A, B, and C. This megakaryocyte had a dense-appearing nucleus, platelet pseudopodia at the peripheral boundaries of the cell, and scant hypogranular cytoplasm. The striking abnormality in these megakaryocytes was the finding of erythrophagocytosis of mature erythrocytes. These erythrocytes often appeared to be enclosed within a vacuolar structure in the cytoplasm, as seen in Figure 3, A, B and C. A fifth type of abnormal megakaryocyte is seen in Figure 4, A and B. This reticuloid megakaryocyte 3 was frequently smaller than the normal megakaryoblast, and demonstrated a finely-reticular nuclear chromatin network and scant, hypogranular cytoplasm. The cell was fre-
FIG. 2. A, megakaryocyte with numerous dense aggregates of nuclear chromatin and doughnut hole in center. The cytoplasm is scant and hypogranular. B, megakaryocyte with similar doughnut hole and several bulbous-appearing nuclear lobules. The cytoplasm is scant and contains few granules. C, megakaryocyte with eccentrically located nucleus and multiple cytoplasmic vacuoles. Abortive platelet formation can be seen at the periphery of the cell. D, megakaryocyte with similar eccentric nucleus and multiple cytoplasmic vacuoles of various sizes. Vacuole-filled pseudopodia are seen, and an area of granular cytoplasm is adjacent to the nucleus. Wright's stain, x 1,200. quently binucleate, and platelet formation at the periphery could be seen. A sixth group of megakaryocytes did not possess any common abnormality. Nevertheless, these megakaryocytes showed numerous individual aberrations, including abnormalities of nuclear segmentation and caliber of chromatin strands. These abnormalities are illustrated in Figure 5, A, B and C. A cytochemical stain for LDH activity in the megakaryocytes of the four marrow samples revealed dense cytoplasmic accumulations of formazan granules representing LDH activity (Fig. 6). The number of these granules was considerably greater than in normal or ITP megakaryocytes and resembled the LDH activity seen in essential thrombocythemia and agnogenic myeloid metaplasia. 8
KASS AJ.C.P. Vol. 61 FIG. 3. A, B, and C, megakaryocytes from three different patients with TTP illustrate erythrophagocytosis. Mature erythrocytes, some of which appear partially digested, are found in the cytoplasm (arrows). Wright's stain, x 1,200. FIG..4./4 andb, megakaryocytes from two patients. These small cells have finely reticular nuclear chromatin. Platelet formation can be seen at the periphery of the cell. These megakaryocytes are frequently binucleate. Wright's stain, x 1,200. FIG. 5. A, megakaryocyte with coarsely-fenestrated nuclear chromatin pattern and scant hypogranular cytoplasm. Lobulations are not seen. B, megakaryocyte with three separate nuclei, cytoplasmic vacuolation, and abortive platelet formation at the periphery. C, large young-appearing megakaryocyte^ megakaryoblast). Wrights stain, x 1,200.
May 1974 MEGAKARYOCYTES IN TTP 643 Discussion Although the clinical manifestations of thrombotic thrombocytopenic purpura have been well characterized since its description by Moschowitz in 1925, 7 the etiology of the disorder is still unknown. 1 The results of the present study suggest that the thrombocytopenia of TTP may have at least part of its origin in morphologically abnormal megakaryocytes. The first, or large, type of megakaryocyte resembles those seen in essential thrombocythemia. Increased activity of LDH enzyme has been found in the megakaryocytes of essential thrombocythemia. 5 Since increased activities of glycolytic enzymes are found in neoplastic cells, 9 it has been suggested that the abnormal-appearing megakaryocytes of essential thrombocythemia may be neoplastic. 5 FIG. 6. Megakaryocyte, TTP, LDH stain and nuclear fast red counterstain, showing numerous dark formazan granules representing LDH activity, x 1,200. The present study illustrates such megakaryocytes containing phagocytized erythrocytes. Erythrophagocytosis by reticulum cells is commonly observed in TTP The second type of megakaryocyte, containing dense chromatin aggregates and a "doughnut-hole" nucleus, may also indicate an intrinsic megakaryocyte abnormality. marrows, 6 and may account in part for Although this is speculative, the anemia. It is difficult to assess the the dense chromatin aggregates may be contribution of megakaryocytic erythrophagocytosis caused in part by collections of nucleohistones. These focal collections of histones could repress certain types of genetic information, leading to cytoplasmic hypogranuiarity and decreased platelet formation. The "doughnut-hole" may represent a defect in nuclear division. The third to the development of the anemia of TTP, but perhaps it may play a minor role. The factor(s) making the erythrocyte susceptible to engulfment by the megakaryocyte and the factor(s) which may lead to increased activity of the megakaryocytic cell membrane, causing it to type of megakaryocyte, with an eccentric behave as a phagocyte in TTP, are largely nucleus and multiple cytoplasmic vacuoles, unknown. The fifth, or "reticuloid," group resembles some of the megakaryocytes of megakaryocytes strongly resembles observed in idiopathic thrombocytopenic those seen in myeloproliferative disorders, purpura. 2,3 Such megakaryocytes in the particularly chronic granulocytic leukemia, latter condition are believed to be vacuolated agnogenic myeloid metaplasia, and acute because of "toxic" influences. It is and chronic erythremic myelosis. 3 The iso uncertain whether this is the case in TTP lated nuclear segmentation and chromatin since a "toxin" has not yet been identified. abnormalities in TTP resemble those Although fibrin strands surrounding found in both acute leukemias 3 and idiopathic and penetrating megakaryocytes have been thrombocytopenic purpura. 2,3 observed in TTP, 8 erythrophagocytosis Recent cytochemical studies of proerythroblasts by megakaryocytes has not been described. in TTP 6 have shown that the
644 KASS AJ.C.P. Vol. 61 proerythroblasts contain punctate deposits of arginine-rich histone in a speckled pattern. Among a variety of anemias, only proerythroblasts in the DiGuglielmo syndrome (acute and chronic erythremic myelosis and erythroleukemia) and in TTP were found to have this speckling. 4,6 A megaloblastoid type of erythroid chromatin pattern such as that found in the DiGuglielmo syndrome is also found in the erythroid precursors of TTP. 6 The cytochemical and morphologic similarities between the erythroid precursors of TTP and the DiGuglielmo syndrome suggested the possibility of an intrinsic and perhaps neoplastic erythroid abnormality in TTP. 6 In the present study, most TTP megakaryocytes were found to have markedly elevated LDH activities. Because LDH elevations of this magnitude are commonly found in neoplastic cells, 9 the possibility is raised that the megakaryocytes in TTP may also be neoplastic, and perhaps part of a wider panmyelotic disturbance. The suggestion that TTP may be a neoplastic disorder contrasts with a more widely-accepted belief 1 that TTP may be a type of disseminated intravascular coagulation. According to this consumptivecoagulopathy theory of TTP, the bizarre changes in the megakaryocytes may be the result of the same catastrophic event that has occurred in the microvasculature. References 1. Amorosi EL, Ultmann JE: Thrombotic thrombocytopenic purpura: Report of 16 cases and review of the literature. Medicine 45:139-159, 1966 2. Dameshek W, Miller EB: Megakaryocytes in idiopathic thrombocytopenic purpura; a form of hypersplenism. Blood 1:27-36, 1946 3. Kass L: Bone Marrow Interpretation. Springfield, 111., Charles C Thomas, 1973 4. Kass L: Demonstration of histones in proerythroblasts in pernicious anemia and the Di Guglielmo syndrome. J Histochem Cytochem 20:817-820, 1972 5. Kass L: Enzymatic abnormalities in megakaryocytes. Acta Haematol 49:193-199, 1973 6. Kass L: Nuclear "speckling" in thrombotic thrombocytopenic purpura proerythroblasts. Am J Clin Pathol 59:869-871, 1973 7. Moschowitz E: An acute febrile pleiochromic anemia with hyaline thrombosis of the terminal arterioles and capillaries. An undescribed disease. Arch Intern Med 36:89-93, 1925 8. Rebuck JW, Boyd CB, Monto RW: Morphologic evaluation of megakaryocytes, The Blood Platelets. Baltimore, Williams and Wilkins, 1971, pp 26-53 9. Shonk CE, Arison RN, Koven BJ, et al: Enzyme patterns in human tissues. III. Glycolytic enzymes in normal and malignant tissues of the colon and rectum. Cancer Res 25: 206-214, 1965 10. Stuart J, Skowron PN: A cytochemical study of marrow enzymes in megaloblastic anaemia. Br J Haematol 15:443-454, 1968