Acta neuropath. (Berl. 36, 327--338 (1976)

Acta Neuropathologica 9 by Springer-Verlag1976

Lymphocyte Inclusions in the JuvenileType of Generalized Ceroid-Lipofuscinosis An Electron Microscopic Study Gtinther Schwendemann Department of Neuropathology and Experimental Brain Research, Universit~its-KrankenhausEppendorf, Martinistr. 52, D-2000 Hamburg 20, Federal Republic of Germany

Summary. Peripheral blood lymphocytes exhibiting cytoplasmic vacuoles from eight patients with the juvenile type of generalized ceroid-lipofuscinosis were investigated by electron microscopy. Osmiophilic inclusions could be demonstrated in the vacuoles and occasionally in the cytoplasm proper, revealing a fingerprint-like ultrastructural pattern or a less defined dense granular material, sometimes with an osmiophobic component adjoined. The inclusions are compared with those demonstrated in seven of the cases by earlier investigations of rectal biopsies in a variety of cell types, mainly in nerve cells. Discrimination is attempted between the lymphocyte inclusions and "parallel tubular arrays" regarded as representing a regular lymphocyte constituent. INTRODUCTION As a characteristic sign in the Spielmeyer-Sj6gren type of the Batten-Vogt syndrome (juvenile form of so-called neuronal ceroid-lipofuscinosis or, more accurately, generalized ceroid-lipofuscinosis--GCL) rendering diagnostic significance, lymphocytes with cytoplasmic vacuoles were first demonstrated light microscopically by von Bagh and Hortling [4] who reported about six cases in Finland. This has been confirmed by further observations in Sweden [41], Norway [36], France [53], Denmark [38, 51], Brazil [28], Germany [12], and U.S.A [47]. Clinically non-affected relatives, thought to be heterozygous for the strain, may likewise reveal vacuolized lymphocytes, mostly in lower percentages [42]. The percentages given in affected cases vary in a wide range from 0.5 ~ [29] up to 77 ~ [36], with a high fluctuation in individual cases counted at different times [39] and with or without vacuolized lymphocytes in stages of the disease prior to exhibiting ophthalmological and neurological symptoms [39]. The dependency of their number on the duration of the disease has been affirmed [4,46] or questioned [12]. Cases revealing no lymphocyte vacuoles have also been reported [23]. Moreover, vacuolized lymphocytes are by no means specific for the juvenile type 23

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of GCL. Irrespective of quite different pathological conditions, such as lymphoid leukemias, toxic situations, infectious diseases, or various diseases of the central nervous system unrelated to lipidoses [12,46,48], they may be seen not only in the late-infantile type of GCL (Jansky-Bielschowsky) [12], but also in Guzz-gangliosidoses (Tay-Sachs) [47], in Gm-gangliosidoses (Norman-Landing)[37], in sphingomyelinoses (Niemann-Pick) [1 ], in mucopolysaccharidoses [34] as well as in various other "storage diseases" (for literature, see Heyne et al. [22]). Hence, early electron microscopical investigations were undertaken to obtain further details on the vacuolized lymphocytes in the Spielmeyer-Sj6gren syndrome, particularly since histochemical studies either failed to reveal an affinity of the lymphocyte vacuolar content for various lipid stains [4, 12, 14, 28, 35, 39, 53] and PAS stain [14, 28, 35] or, if lipid solvents in fixation were avoided, they have shown positive results in some of the vacuoles, while revealing identical or similar findings in other lipidoses [46, 48]. Yet, with procedures available at that time, no electron-dense material could be detected within the lymphocyte vacuoles [30, 39]. Later on, Fukuda and Miura [14] were able to demonstrate electron microscopically membranous cytoplasmic bodies--as originally depicted within neuronal cytoplasm in Tay-Sachs disease [52]--as well as cytoplasmic bodies which were partly filled with electron-dense, amorpos or parallel double membrane-like structures, probably corresponding to lymphocytic vacuoles. Furthermore, Nasu et al. [35] observed various inclusions within the vacuoles, partly with lamellar, reticulated or membranous structures, and Witzleben et al. [58] and Witzleben [57] described inclusions ranging from "dense, apparently homogenous, osmiophilic inclusions to laminar and apparent tubular profiles", mostly within cytoplasmic vacuoles, but also free in the cytoplasm. However, the inclusions described by Witzleben et al. [58] have been argued to be identical with lymphocyte cytoplasmic "tubule-containing vacuoles (TCV)" [2]. In our opinion, some of the inclusions depicted by Nasu et al. [35] likewise seem to be identical with these TCV. The authors had already mentioned the occurrence of partly similar structures in lymphocytes of normal human blood. These structures, which have been defined by various synonyms, e.g. "parallel tubular arrays (PTA)" [10,17], "intracytoplasmic tubular bodies" [43], "parallel tubular structures" [20] or merely "tubular structures" [25], in different pathological conditions as well as normal situations, probably represent a true lymphocytic organelle [10,25,56]. They should be properly distinguished from tubuloreticular structures [2,17,10, 20] seen in lymphocytes and other cells, which were once linked to virus infection [5,18]. Based on eight cases with juvenile type of GCL, we want to report further electron microscopical investigations aimed at finding out whether there are inclusions in the vacuolized lymphocytes of peripheral blood related to the disease and distinguishable from the TCV lymphocytic constituent. CLINICAL DATA AND METHODS Venous blood was drawn from eight children or adolescents, considered to suffer from the juvenile type of GCL. Blood from two apparently normal children and two adults as well as from three children affected with diseases unrelated to lipidoses was likewise

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examined and served as control. The diagnosis ' G C L ' was established in seven cases by the light and electron microscopical investigation of a rectal biopsy and of an additional sural nerve biopsy in four of the seven, the remaining case being the sister of one of them. A report of the bioptical findings has been given elsewhere [45]. Classification of the juvenile form of G C L was done according to the clinical course and symptoms as defined by Zeman et al. [59]. The cases are characterized by an early manifestation of visual disturbances, registered as an initial sign between ages 4 and 7 years in seven of the cases. In one case grand real seizures are reported to have occurred at age 6 years, mental disabilities were noticed after one year and advanced ophthalmological alterations were documented 3 years later. At the time of the lymphocyte investigation, the duration of the disease had been from 5 to 13 years, all patients being still alive. In two of them, seizures had not yet been noticed but had been registered at early or later stages in the remaining cases. The seizure activity was low except in far advanced stages. The percentage of lymphocytes revealing cytoplasmic vacuoles was determined in blood smears in seven of the cases to be ranging from 6% to 31%; in one case it was estimated in about the same order of percentage. F o r the electron microscopical investigation of the lymphocytes white blood cells were isolated from the venous blood specimens by the isolation technique according to B6yum [9], which were kindly processed for us by the laboratories of the UniversitfitsKinderklinik, Hamburg, with a modification introduced by Dr. K. Winkler. F o r fixation, the isolated leukocytes were suspended in 3 ~ phosphate-buffered glutaraldehyde solution and gently centrifuged (less than 700 rpm) for 15 min. The sedimented leukocytes were resuspended for 2 h in fresh glutaraldehyde solution. Centrifugation was mostly unnecessary in the following steps, the leukocytes aggregating to small clots when suspended for postfixation in Dalton's chrome-osmium solution for 2 h. After dehydration with graded alcohol and propylenoxide, the leukocytes were embedded in Epon 812, settling on the bottom of gelatin capsules; semithin sections were stained with toluidine-blue for orientation and ultrathin sections were cut with a Reichert OmU2, poststained with uranyl acetate and lead citrate, and investigated with a Zeiss EM 9S electron microscope. F o r comparison, additional venous blood specimens both from cases with G C L and from controls were processed for 'buffy coat' lymphocytes by established methods, the solutions for fixation and postfixation, dehydration, embedding and poststaining corresponding to those just described. No essential differences were noticed between these two methods with regard to the quality of fixation and the ultrastructural findings.

RESULTS A s seen with the electron m i c r o s c o p e , the l y m p h o c y t e c y t o p l a s m i c vacuoles are limited b y unit m e m b r a n e s m e a s u r i n g up to 2 ~zm in d i a m e t e r (Figs. 1 - - 6 ) . C o n n e c t i o n o f the limiting m e m b r a n e with those o f the e n d o p l a s m i c reticulum has never been verified. M a n y o f the vacuoles a p p e a r " e m p t y " , revealing p r a c t i c a l l y no electron-dense inclusions with the p r o c e d u r e s a p p l i e d (Fig. 1 A). This c e r t a i n l y d e p e n d s also on the section plane, b u t c o u l d be ascertained in a few instances b y serial sections. O t h e r vacuoles exhibit only an i r r e g u l a r floccular m a t e r i a l o f m o d e r a t e electron density (Fig. 2). A further n u m b e r o f vacuoles, however, d i s p l a y a dense, o s m i o p h i l i c m a t e r i a l , filling t h e m to a v a r y i n g degree. I n this m a t e r i a l an u l t r a s t r u c t u r a l p a t t e r n m a y be resolved with high magnifications (Figs. 1--3). This p a t t e r n consists o f p a r a l l e l pairs o f electron-dense lamellae, the lamellae being a b o u t 2 0 - - 2 5 A thick with a clear zone o f a b o u t 1 0 - - 1 5 A between the two p a i r e d lamellae. The distance 23*

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Fig. 1. (A) Lymphocyte with membrane-bounded cytoplasmic vacuoles, appearing "empty" in the section plane or filled with osmiophilic inclusions to a varying degree, • 8000. The inclusion of the left vacuole reveals a fingerprint-like pattern with higher magnification (B), • 93000

Fig. 2. (A) Cytoplasmic vacuoles of a lymphocyte containing irregular floccular material and an additional osmiophilic inclusion (left upper vacuole), • 19000. The latter (B) exhibits fingerprint-like profiles (arrow). A less defined inclusion with reduced electron contrast is adjoined (crossed arrow), • 83 000 between the pairs measures about 20--30 ,~. The lamellae are straight or slightly curved. On occasion, a second system of paired lamellae seems to be interposed at an angle, resulting in a crystalloid lattice (Fig. 3 B). In many instances, however, probably also depending partly on the section plane and conditions of fixation,

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Fig. 3. (A) Lymphocyte vacuole containing osmiophilic inclusions with a fingerprintlike pattern (arrows) and a lipid droplet-like spheroid of varying electron density associated (crossed arrow), • 56000. Higher magnification (B) reveals parallel paired lamellae, partly with a crystalloid lattice by a second system of paired lamellae interposed at an angle (arrow), x 135000

even with high resolution no linear arrays can be seen within the osmiophilic inclusions or parts of them; they merely present a dense, more or less granular (Fig. 4) or almost homogeneous material. Furthermore, the osmiophilic deposits are occasionally found to display a somewhat reduced electron contrast, either in the total deposit or in parts (Figs. 5 and 6). In the latter case, this is likely to be seen in the deposit center which is surrounded by a rim of more osmiophilic material (Fig.6). From this, gradual steps may be pursued to see pictures which are reminiscent of the droplet-like osmiophobic component seen in lipopigments (Figs.3A and 6B). The electrondense vacuole inclusions may supplementarily be adjoined by the floccular material mentioned above (Figs.2, 5 and 6). As a rule, the electron-dense inclusions just described are found within the lymphocyte cytoplasmic vacuoles. Nevertheless, as an occasional finding in our cases, corresponding deposits may be detected within the lymphocyte cytoplasm proper, without or only with fragments of a limiting membrane (Fig. 7). Although tangentional sectioning as well as artifacts should always be considered, one might speculate that these represent developmental stages of the vacuolar inclusions. No comparable lymphocytic inclusions were detected in the blood specimens examined as controls. However, in the controls as well as in each of the cases with GCL investigated, a low percentage of peripheral blood lymphocytes revealed PTA (Fig.8A and B). The tubules are 250--300 A in diameter, their walls being about 40--60 A thick. Individual tubules may be found loosely arranged, displaying an irregular course, but mostly the tubules are in wall-to-wall contact, their course being parallely straight or wounded. Some dense amorphous material may be found adjoined (Fig.8B). The tubules may be seen "free" in the cytoplasm or partly or totally bounded by a limiting membrane, the latter instances

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Fig. 4. Lymphocyte vacuole containing a dense granular material. In addition, some short fingerprint-like profiles are seen (arrow), • Fig. 5. (A) Lymphocyte vacuoles with floccular material, an electron-dense inclusion (arrow), and with inclusions with reduced electron contrast (crossed arrow), • 10000. (B) A faint fingerprint-like pattern is discernible in peripheral portions of the latter (arrows), • 83000 Fig. 6A and B. Lymphocyte vacuoles with inclusions with droplet-like centers surrounded by rims of dense material; (A) • 10000, (B) • 60000

resulting in cytoplasmic TCV. Nota bene, PTA or TCV may additionally be seen in lymphocytes, displaying cytoplasmic vacuoles with or without the various inclusions described above. On rare occasions, PTA, usually those that are non-membrane bounded, may be detected associated with some of the parallel paired osmiophilic lamellae.

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Fig. 7. Hypothetical developmental stages in lymphocyte inclusion development. No limiting membrane is discernible (arrow) bounding the osmiophilic inclusion with fingerprint-like profiles in the right upper corner, • 90000

Fig. 8. (A) PTA within lymphocyte cytoplasmic vacuoles (arrows), • 34000. (B) PTA consisting of tubules apposed wall to wall (arrows). Some dense amorphous material is adjoined (crossed arrow), • 105000 DISCUSSION As mentioned initially, PTA or TCV are likely to represent a regular lymphocyte constituent, even though they may be found more frequently in some pathological instances and/or certain lymphocyte developmental stages [2,55,56], recently

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argued to be most prominent in T-lymphocytes [10]. Thus it is not surprising that we have also seen them in the lymphocytes in each of our cases, their ultrastructure corresponding exactly to that documented in the literature [24--27]. From this lymphocyte constituent, the various inclusions in vacuolized lymphocytes from cases with the juvenile type of GCL as reported above are to be distinguished. With appropriate resolving conditions, the pairs of lamellae are separated by clear distances of about 20--30 A and display a light line of about 10--15 A in the middle of each pair, whereas in PTA tubules with a light diameter of about ]50--200 A are in apposition. Particularly in those rare instances in which PTA and parallel pairs of lamellae are seen associated, again both structures are properly to be distinguished by their different profile patterns and dimensions. Considering the lymphocyte inclusions with a pattern of parallel paired lamellae, there is a striking ultrastructural similarity to that of the various lipopigment patterns described in GCL, referred to as fingerprint-like [49]. Indeed, in seven of the eight cases presented, with additional investigation of rectal biopsies, we have been able to demonstrate inclusions with a fingerprint-like pattern also in the perikarya of myenteric nerve cells, both within cytoplasmic vacuoles and within the cytoplasm proper [45]. These inclusions are nltrastructurally very similar to those found in the lymphocytes, in the qualified sense that they reveal slightly wider distances between the lamella pairs (about 30--60 A). Inclusions with a fingerprint-like pattern which are morphologically identical with those in lymphocytes were seen in each of the rectal biopsies in the cytoplasm of various nonneuronal cell types, e.g. endothelial cells and pericytes, smooth muscle cells, histiocytes, fibroblasts, myenteric plexus Schwann cells, and even plasma cells. However, each of these non-neuronal cell types (though not the intramural nerve cells, with a certain exception in one case) displayed in addition deposits with another pattern which were frequently combined within an individual lipopigment granulel In the literature, the pattern of these deposits is preferentially referred to as "curvilinear profiles" [13]. No such distinct curvilinear profiles could be detected in the lymphocytes investigated. AS previously argued, the type of cell may probably to some degree determine the final architecture of the deposits in GCL [15,45]. On the other hand, some of the classifications proposed in GCL were based on the predominance of a certain ultrastructural pattern of lipopigment. Thus, granular osmiophilic bodies were shown to be prevalent in distinct types [8,11,21]. In our cases, which according to clinical criteria were classified as belonging to the juvenile type of GCL (Spielmeyer-Sj6gren), granular osmiophilic material could be seen isolated or combined with different patterns of deposits in lymphocytes as well as in the nerve cells and the various non-neuronal cells of the rectum. Even though in GCL, which affects mainly the central nervous system, there is no clinical evidence for a generalized systemic disease, visceral involvement, i.e. an accumulation of lipopigment (ceroid and/or lipofuscin) within non-nervous tissue elements is well documented by light and electron microscopy (for literature, see Zeman et al. [59]). However, as far as the deposits in lymphocytes are concerned, their lipopigment nature has not yet been substantiated. Cytochemically, some of the lymphocyte vacuoles revealed in the light microscope material which was positive for lipid stainings and PAS stain [46,48], but PTA are likewise

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faintly PAS-positive and stain slightly with Sudan Black [24,25]. Nevertheless, there remains a striking ultrastructural congruence and the fact that, as proven by light and electron microscopy, the lymphocytic deposits in contrast to PTA [25] are positive for acid phosphatase activity [35], as are ceroidlipofuscin granules in G C L [16, 31,49], which provides evidence for their lysosomal nature. Despite our own findings, we still hesitate to consider the vacuolized lymphocytes with the inclusions described as a specific diagnostic marker in GCL. Curvilinear profiles, so far thought to be most characteristic in this disease, were not discernible. Fingerprint-like lamellae, however, are by no means uniquely found in GCL. Irrespective of the various pathological as well as normal situations in animals, they have been described in human beings in quite different pathological [7,19,54,59] and even normal [44] conditions as well as (supplementarily) in various lipidoses apart from G C L [3, 32, 50]. Nevertheless, recurring to the inclusions seen with the electron microscope in peripheral blood lymphocytes of patients with the juvenile type of G C L - - i n fact, in each of our cases at a considerable percentage--no identical inclusions have been described in vacuolized lymphocytes in the different "storage" disorders on which, admittedly, relatively few reports are available [6, 22, 33, 40].

Acknowledgements. The author gratefully acknowledges the cooperation of Dr. K.-L. Elze, Allgemeines Krankenhaus Altona, Department of Ophthalmology, Hamburg, and Dr. H.-P. Koepp, Universit/its-Kinderklinik, Hamburg (Director: Prof. K.-H. Schfifer). He wishes to thank Mrs. K. Stroth and Miss C. Weissleder for expert technical assistance. REFERENCES 1. Abt, A.F., Bloom, W.: Essential lipoid histiocytosis (type Niemann-Pick). A congenital disturbance in lipoid metabolism in infants associated with a splenohepatomegaly. J. Amer. reed. Ass. 90, 2076--2080 (1928) 2. Anzil, A.P., Blinzinger, K.: Cytoplasmic tubule-containing vacuoles and endoplasmic tubuloreticular inclusions in the lymphocytes of a child with an unidentified form of cerebroretinal degeneration. Biomedicine 21, 210--2t2 (1974) 3. Anzil, A. P., Blinzinger, K., Mehraein, P., Dozic, S. : Niemann-Pick disease type C: Case report with ultrastructural findings. Neuropfidiatrie 4, 207--225 (1973) 4. Bagh, K. v., Hortling, H. : Blodfynd vid juvenil amaurotisk idioti. Nord. Med. 38, 1072--1076 (1948) 5. Bariety, J., Amor, B., Kahan, A., Balafrej, J.L., Delbarre, F.: Ultrastructural anomalies in mononuclear cells of peripheral blood in S. L. E. : Presence of viruslike inclusions. Rev. europ, t~tud, clin. biol. 16, 715--720 (1971) 6. Belcher, R. W. : Ultrastructure and cytochemistry of lymphocytes in the genetic mucopolysaccharidoses. Arch. Path. 93, 1--7 (1972) 7. Biava, C., West, M. : Lipofuscin-like granules in vascular smooth muscle and juxtaglomerular cells of human kidneys. Amer. J. Path. 47, 287--3t4 (1965) 8. Boehme, D. H., Cottrell, J. C., Leonberg, S. C., Zeman, W.: A dominant form of neuronal ceroid-lipofuscinosis. Brain 94, 745--760 (1971) 9. B6yum, A. : Isolation of leucocytes from human blood. A two-phase system for removal of red cells with methylcellulose as erythrocyte-aggregating agent. Scand. J. clin. Lab. Invest. 21, Suppl. 97, 9--29 (1968) 10. Brunning, R. D., Parkin, J. : Ultrastructural studies of parallel tubular arrays in human lymphocytes. Amer. J. Path. 78, 59--70 (1975)

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11. Carpenter, S., Karpati, G., Wolfe, L. S., Andermann, F. : A type of juvenile cerebromacular degeneration characterized by granular osmiophilic deposits. J. neurol. Sci 18, 67--87 (1973) 12. Derwort, A., Detering, K. : Vacuolisierte Lymphocyten bei familifirer amaurotischer Idiotie und ihre diagnostische Bedeutung. Nervenarzt 30, 442 448 (1959) 13. Duffy, P. E., Kornfeld, M., Suzuki, K.: Neurovisceral storage disease with curvilinear bodies. J. Neuropath. exp. Neurol. 27, 3 5 1 - 3 7 0 (1968) 14. Fukuda, K., Miura, A. B.: The ultrastructure of vacuolated lymphocytes in the peripheral blood of juvenile amaurotic familial idiocy. Tohoku J. exp. Med. 89, 113--120 (1966) 15. Goebel, H. H. : The ultrastructural spectrum of residual bodies in neuronal ceroidlipofuscinosis. In: Proc. VIIth Int. Congress Neuropath. Budapest 1974. Vol. 1, pp. 249--252. Amsterdam: Excerpta Medica 1975 16. Gonatas, N. K., Gambetti, P., Baird, H. : A second type of late infantile amaurotic idiocy with multilamellar cytosomes. Path. europ. 3, 323--331 (1968) 17. Goodman, J. R., Sylvester, R. A., Talal, N., Tuffanelli, D. L. : Virus-like structures in lymphocytes of patients with systemic and discoid lupus erythematosus. Ann. intern. Med. 79, 396--402 (1973) 18. Grimley, P. M., Decker, J. L., Michelitch, H. J., Frantz, M. M. : Abnormal structures in circulating lymphocytes from patients with systemic lupus erythematosus and related diseases. Arthr. and Rheum. 16, 313--323 (1973) 19. Gy6rkey, F., Shimamura, T., O'Neal, R. M. : The fine structure of ceroid in human atheroma. J. Histochem. Cytochem. 15, 732--736 (1967) 20. Halie, M. R., Splett-Romascano, M., Molenaar, I., Nicweg, H. O. : Parallel tubular structures in lymphocytes. I. Occurrence in patients with Hcdgkin's disease. Acta haemat. (Basel) 54, 18--26 (1975) 21. Haltia, M., Rapola, J., Santavuori, P. : Infantile type of so-called neuronal ceroidlipofuscinosis. Histological and electron microscopic studies. Acta neuropath. (Berl.) 26, 157--170 (1973) 22. Heyne, K., Kemmer, C., Simon, C., Trtibsbach, A. : Generalisierte GM~-Gangliosidose: Feinstruktur und differentialdiagnostische Bedeutung speichernder Lymphozyten und Knochenmarkszellen. Pfidiat. P/idol. 8, 272--283 (1973) 23. Hoffman, J. : Pigmentary retinal lipoid neuronal heredodegeneration (SpielmeyerVogt disease). Further observations on the presence of vacuolized lymphocytes. Acta psychiat, scand. 32, 450--456 (1957) 24. Hovig, T., Jeremic, M., Stavem, P. : A new type of inclusion bodies in lymphocytes. Scan& J. Haemat. g, 81--96 (1968) 25. Huhn, D.: Neue Organelle im peripheren Lymphozyten? Dtsch. med. Wschr. 44, 2099--2100 (1968) 26. Huhn, D., Tympner, K.-D.: Elektronenoptische Untersuchungen der Lymphozyten bei verschiedenen Formen von Antik6rpermangel im Kindesalter. Blut 20 169--177 (1970) 27. Jorke, D. : Die submikroskopische Struktur einer neuen zytoplasmatischen Organelle in Lymphozyten und Lymphoidzellen. Folia haemat. (Lpz.) 94, 1 --10 (1970) 28. Juligo, O.F., Canelas, H . M . , Longo, N. A. : Idiotia amar6tica familiar, forma juvenil. Estudo clinico e laboratorial de tr~s casos. Arch. Neuro-psiquiat. (S. Paulo) 14, 136--157 (1956) 29. Kivalo, A., Kivalo, E. : Juvenile amaurotic idiocy. Vacuolisation of lymphocytes in the healthy members of families involved. Ann. Paediat. Fenn. 4, 191--195 (1958) 30. Kivalo, E., StjernvaI1, L. : Vacuolized lymphocytes in juvenile amaurotic idiocy. An electron microscopic study. Ann. Paediat. Fenn. 4, 25--29 (1958) 31. Koenig, H., McDonald, T., Nellhaus, G. : Morphological and histochemical studies of neurolipidosis by light and electron microscopy. J. Neuropath. exp. Neurol. 23, 191 (1964)

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32. Kornfeld, M., Appenzeller, O., Saiki, J., Troup, G. M.: Sea-blue histiocytes and sural nerve in neuroviscerat storage disorder with vertical ophthalmoplegia. J. neurol. Sci. 25, 291 --302 (t975) 33. Lazarus, S. S., Vethamany, V. G., Schneck, L., Volk, B. W.: Fine structure and histochemistry of peripheral blood cells in Niemann-Pick disease. Lab. Invest. 17, 155 --170 (1967) 34. Mittwoch, U.: Abnormal lymphocytes in gargoylism. Brit. J. Haemat. 5, 365 368 (1959) 35. Nasu, T., Kitani, T., Imanaka, T., Yonezawa, T., Enomoto, T. : Vacuolated lymphocyte in a case of juvenile amaurotic family idiocy. Acta haemat, jap. 32, 443 --455 (1969) 36. Nissen, A. J.: Juvenil amaurotisk idioti in Norge. Nord. Med. 52, 1542--1546 (1954) 37. Norman, R. M., Urich, H., Tingey, A. H., Goodbody, R . A . : Tay-Sachs' disease with visceral involvement and its relationship to Niemann-Pick's disease. J. Path Bact. 78, 409--421 (1959) 38. Plum, C. M.: Lymphocyte degeneration in amaurotic idiocy. Dan. med. Bull. 4, 156-- 157 0957) 39. Plum, C.M., Teglbjaerg, H. P. S. : Juvenile amaurotic idiocy. Vacuolisation of lymphocytes. Ann. Paediat. Fenn. 6, 16 20 (1960) 40. Rapola, J., Autio, S., Aula, P., Nanto, V. : Lymphocytic inclusions in I-cell disease. J. Pediat. 85, 88--90 (1974) 41. Rayner, S.: Juvenile amaurotic idiocy. Diagnosis of heterozygotes. Acta genet. (Basel) 3, 1 5 (1952) 42. Rayner, S. : Juvenile amaurotic idiocy in Sweden. With particular reference to the occurrence of vacuoles in the lymphocytes of homo- and heterozygotes. Lund: Ohlssons Boktryckeri 1962 43. Reyes, F., Le Go, A., Delrieu, F., Bach, J. F. : Ultrastructure of cells binding immunoglobulin-coated erythrocytes in rheumatoid arthritis. Clin. exp. Immunol. 17, 5 3 3 - 5 4 6 (1974) 44. Schlote, W., Boellaard, J . W . : Alterskorrelierter Strukturwandel des neuronalen Lipopigments beim Menschen. Verb. dtsch. Ges. Path. 59, 304--309 (1975) 45. Schwendemann, G., Colmant, H. J.: Bioptic studies on ceroid-lipofuscinosis: Light and electron microscopic observations. In: Proc. VIIth Int. Congress Neuropath., Budapest 1974. Vol. 1, pp. 209 212. Amsterdam: Excerpta Medica 1975 46. Spiegel-Adolf, M., Baird, H. W., Coleman, H. S., Szekely, E. G. : Vacuolized blood lymphocytes in the lipidoses and other central nervous system diseases with special reference to histochemical studies. In: Cerebral sphingolipidoses (eds. S. M. Aronson and B. W. Volk), pp. 129--140. New York-London: Academic Press 1962 47. Spiegel-Adolf, M., Baird, H. W., Kollias, D., Szekely, E. G. : Cerebrospinal fluid, serum, and blood investigations in amaurotic family idiocy. Amer. J. Dis. Child. 97, 676-683 (1959) 48. Spiegel-Adolf, M., Baird, H . W . , Szekely, E.G., Coleman, H. S. : Vacuolized lymphocytes in CNS diseases with special reference to amaurotic familial idiocy Confin. neurol. (Basel) 20, 343--354 (1960) 49. Suzuki, K., Johnson, A. B., Marquet, E., Suzuki, K.: A case of juvenile lipidosis: Electron microscopic, histochemical and biochemical studies. Acta neuropath. (Berl.) 11,122--139 (1968) 50. Taori, G. M., Basu, D. K., Chandi, S., Raman, P. T., Abraham, J., Leelavathy, R., Job, C. K. : GM1 Gangliosidosis. J. neurol. Sci. 21, 7 7 - 9 2 (1974) 51. Teglbjaerg, H. P.S., Plum, C. M. : Vacuolized lymphocytes in lipoidosis. Acta psychiat, scand. 30, 327--341 (1955) 52. Terry, R. D., Korey, S. R. : Membranous cytoplasmic granules in infantile amaurotic idiocy. Nature (Lond.) 188, 1000--1002 (1960)

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53. Thi6baut, F., Waitz, R., Rohmer, F., Brini, A., Israel, L. : Un cas de forme juv6nile de l'idiotie amaurotique type Spielmeyer-Vogt. Rev. neurol. 90, 235--238 (1954) 54. Tsukahara, Y., Nasu, T. : Ceroid-like pigment in age changes of human cartilage. Acta path. jap. 24, 357--369 (1974) 55. White, J. G. : Giant organelles containing tubules in Chediak-Higashi lymphocytes. Amer. J. Path. 69, 225--238 (1972) 56. White, J. G. : Lymphocyte inclusions. Ann. intern. Med. 76, 1042--1043 (1972) 57. Witzleben, C. L. : Lymphocyte inclusions in late-onset amaurotic idiocy. Value as a diagnostic test and genetic marker. Neurology (Minneap.) 22, 1075--1078 (1972) 58. Witzleben, C.L., Smith, K., Nelson, J.S., Monteleone, P.L., Livingston, D. : Ultrastructural studies in late-onset amaurotic idiocy: Lymphocyte inclusions as a diagnostic marker. J. Pediat. 79, 285--293 (1971) 59. Zeman, W., Donahue, S., Dyken, P., Green, J. : The neuronal ceroid-lipofuscinoses (Batten-Vogt syndrome). In: Handbook of Clinical Neurology (eds. P. J. Vinken and G. W. Bruyn), Vol. 10, pp. 588--679. Amsterdam: North-Holland Publ. 1970

Received April 26, 1976; Accepted May 10, 1976