Tohoku J. exp. Med., 1975 , 117, 257-281 Electron Microscopic Observation of Inclusion Bodies in Plasma Cells of Multiple Myeloma and Waldenstrom's Macroglobulinemia KOUSUKE OIKAWA Department of Internal Medicine,* Tohoku University School of Medicine, Sendai OIKAWA, K. Electron Microscopic Observation of Inclusion Bodies in Plasma Cells of Multiple Myeloma and Waldenstrom's Macroglobulinemia . Tohoku J. exp. Med., 1975, 117 (3), 257-281 Intracellular inclusion bodies in the plasma cells were sought by electron microscopy in 32 cases of multiple myeloma and 3 of Waldenstrom's macroglobulinemia. In some cases, round shaped intranuclear inclusions and intranuclear fibrillar bundles were observed. In other cases, Russell bodies and crystalline structures were found in the cisternae of rough surfaced endoplasmic reticulum (rER), and dense bodies, myelin-like structures, fibrillar formations, polysome lamellae complexes, crystalline structures, virus-like particles and phagocytosis were observed in the cytoplasm of plasma cells. The detailed ultrastructure of these inclusions was described, and their functional significance, origin and appearance rate were discussed. Finally, the presence of true viral particles in the plasma cells was ruled out. --------- ultrastructure of inclusion bodies; multiple myeloma; Waldenstrom's macroglobulinemia Since the fine structure of the plasma cell was first described by Braunsteiner et al. (1953), many workers have defined the ultrastructure of plasma cell organelles and studied their functions. Furthermore, a variety of intracellular inclusions have been described in the plasma cells of patients with multiple myeloma and Waldenstrom's macroglobulinemia by many authors as reviewed by Maldonado et al. (1966), Waldenstrom (1970), Tanaka and Goodman (1972), Azar and Potter (1973). Little is known, however, about functional significance and origins of these various inclusion bodies; and the studies concerning their appearance rate in plasma cells have not been completed. The purposes of the present study are to describe the ultrastructure of such inclusion bodies, to inquire into their functional signifi- cance and origin, to describe their appearance rate and to check if viral particles are truly present in the plasma cells. Received for publication, August 5, 1975. * Director: Prof. K. Yoshinaga. Present address: The Department of Internal Medicine, Furukawa City Hospital, Furukawa. 257 258 K. Oikawa MATERIALS AND METHODS The specimensof bone marrowwere obtainedby sternal or iliac puncture from untreated14 patientswith IgG myeloma, 7 with IgA myeloma,1 with IgD myeloma, 10 with BJ myelomaand 3 with Waidenstrom'smacroglobulinemia. They were fixed in 2.5% glutaraldehydewith subsequentfixation in 2% osmiumtetroxide, dehydrated in a graded seriesof acetonand embeddedin Epon mixture. Ultrathinsections were stained with uranyl acetateand lead citrate, and examinedwith a Hitachi7S or a JEM 100Ctype electronmicroscope. In each case,at least 200tumor cellswere observed in thin sections. Whenpartic- ular inclusionswere found, several hundreds of cellswere investigated. RESULTS Frequency of appearance of various types of inclusions is shown in Table 1. Round shaped inclusions and fibrilar bundles were observed in the nucleus, Russell bodies and crystalline structures were observed in the cisternae of rough surfaced endoplasmic reticulum (rER), and dense bodies, myelin-like structures, fibrillar formations, polysome lamellae complexes, crystalline structures, virus-like particles and phagocytosiswere found in the cytoplasm of plasma cells. The detailed ultrastructure of these inclusions is described below. Round shaped inclusionsin the nucleus. Intranuclear round shaped inclusions were classifiedinto two types. One had low electron density and contained finely granular material. The other contained strongly osmiophilichomogeneous material similar to Russell body. The first type of inclusion of low electron density was observedin 3 patients, 1 with IgA myeloma (in 22% of plasma cells of Case 16)and 2 with macroglubulinemia(in 8% of plasma cells of Case 35 and less than 1% of plasma cells of Case 34) (Fig. 1). These inclusions were round to oval and their number was usually one, but occasionallytwo to five within a singlenucleus. The size of inclusion bodies varied from about 100 nm in diameter to a huge one of over a few ,um occupying most part of the nucleus. The inclusions were surrounded by a single membrane approximately 70 A thick. The second type of inclusion of high electron density was observed in 2 cases, 1 of IgG myeloma (in less than 1% of plasma cells of Case 5) and 1 of IgA myeloma (in 23% of plasma cells of Case 21). In these cases, one or several (up to ten or more) spherules of different sizes were seen within a single nucleus (Fig. 2). Sometimes, similar structures were noted both in perinuclear cisternae and in cisternae of rER in a single cell (Fig. 3). Occasionally,the inclusion contained the materials character- istic of cytoplasmicstructures such as endoplasmicreticulum and ribosomes(Fig. 4). Both of the above two types of inclusions, whether of low electron density or high density, had neither crystalline nor periodic structure within them. Fibrillar bundlesin the nucleus. Bundlesof fibrilsin the nucleus were observed in less than 1% of plasma cells from 3 patients (in Cases 21, 23, 26). Thesefibrils were always found in the interchromatin space and were arranged in a parallel fashion forming a bundle varying in length and about 200 nm in diameter (Fig. Inclusion Bodies in Plasma Cells of M ultiple Myeloma 259 5a and b). The fibrils had a diameter of a pproximately70 A. A single bundle consisted of dozens of fibers . There was no detectable connection between these structures and nuclear bodies ,nucleoli, nuclear envelope or intracytoplasmic fibrillar formation. Russell body. We observed the Russell bodies in only 3 of 35 cases. In a case of 1gG myeloma(Case 8), the bodies were found in 18% of plasma cells, but in the other 2 cases, 1 of IgA myeloma (Case21) and 1 of BJ myeloma (Case 29), th e bodies were found only in less than 1% of plasma cells. Under the electron microscope, the bodies were found as round and homogeneousmaterial of high electron density within the cisternae of rER (Fig. 6). The sizesof the bodies ranged from about 100 nm to a few microns in diameter. No periodicity could be observed in these structures. Morphologically the bodies were similar to the above-mentioned intranuclear osmiophilic round shaped inclusions. intracisternal crystal. Crystalloid inclusions within the cisternae of rER were observed in 60% of plasma cells of a patient with IgG myeloma (Case 10). In a low magnification, inclusions appeared as many electron dense structures in the shape of needles, spindles or rods (Fig. 7). Under a higher magnification, a longitudinal section showed a marked linearity having periodicity of approximately 100 to 140 A (Fig. 8). In a cross section, geometricallyregular arrangement of tubules of approximately 140 A in diameter, having a central electron lucent core, was observed (Fig. 9). This observation suggested that the inclusions were composed of straight tubular structures measuring 140 A in diameter. Dense body. I have defined the term, dense body, of plasma cell as round to oval osmiophilicstructure within the cytoplasm surroundedby a smooth membrane in order to distinguish it from Russell bodies which are always present within the cisternae of rER. Although the number of dense bodies in plasma cells varied from one case to another, they were found in all 35 cases. In a certain case (Case 18), the bodies were observed in almost all plasma cells. The diameters of the bodies ranged from 0.4 to 1 ,um. Their interior seemed to be dense, faintly granular, and sometimes contained an eccentric dense portion and translucent portion (Fig. 10a), or laminated structures (Fig. 10b). Rarely, intermediate forms between dense bodies and myelin figures were observed (Fig. 10c). Dense bodies were observed near the Golgi area and between the lamellar cisternae; the bodies in the Golgiarea seemed to be lower in density and smallerin size than those in the peripheral cytoplasm (Fig. 11). These observations suggested that the dense bodies were produced in the Golgi area. Myelin-like structure. Myelin-like structures were observed in many plasma cells of approximately half the cases (16 of 35 cases). These structures were present in the cytoplasmic matrix, in mitochondria, in cisternae of rER, in perinuclear cisternae and outside the cells (Fig. 12). Sometimes the picture suggesting that they were being released from the cell was observed (Fig. 13a). 260 K. Oikawa TABLE I. Types of Figures in parentheses are percentage. .c., light chain; R.s., round l shaped; F.b., fibrillar bundle; R.b., Russell body; P.c., polysome lamellae complex; V.p., virus-like particle; Ph., phagocytosis. Inclusion Bodies in Plasma , Cells of Multiple Myeloma 261 inclusions observed Cry., crystal; D.b., dense body; M.s., myelin-like structure; F.f., fibrillar formation; 262 K. Oikawa Under higher magnifications (40,000 or higher), fine parallel striae consisting of light and dense lines of equal width of about 40 to 50 A, running usually in parallel to the edges and forming the broad band, were detected in some places (Fig. 13b). Many of these myelin-like structures were found in the cells considered to be in necrosis, but they were also found in the cells that had no degenerative change in other portions. Fibrillar formation. Cytoplasmic fibrillar formations were observed in 21 of 35 patients. In general, the occurrence of these structures in each case was not very frequent, but occasionally they were noted in as many as 29% of plasma cells (Case 14). Each fibrillar unit measured from about 50 to 70 A in width. The fibrils were arranged in fascicles and/or in a reticular network. The fibrils were distributed randomly in the cytoplasm with some tendency to concentrate near the nuclear membrane and to surround the nucleus (Fig. 14). Sometimes the close association of these fibrils with a nuclear membrane and with mitochondria was noted. Polysome lamellae complex.