Journal of
J. Neurol. 218, 187--196 (1978)
Neurology © by Springer-Verlag 1978
Supplementary Cytodiagnostic Analyses of Mononuclear Cells of the Cerebrospinal Fluid Using Cytological Markers* M. Oehmichen 1 and H. Huber 2 l Institut ftir Hirnforschung der Universit/it Ttibingen (Direktor: Prof. Dr. J. Peiffer), Calwer Strage 3, D-7400 Tfibingen, Federal Republic of Germany 2Medizinische Klinik der Universit/it Innsbruck (Direktor: Prof. Dr. H. Braunsteiner), A-6020 Innsbruck, Austria
Summary. Various methods of CSF cell differentiation are discussed. These methods provide additional information regarding the origin and, therefore, the types of pathological alterations occurring in the leptomeninges. They include the application of immunological and eytochemical markers to differentiate and identify B and T lymphocytes as well as cells from the monocyte-macrophage series and precursor cells of polymorphnuclear leukocytes. Application of these methods to CSF cells and the differentiation of CSF cells from 16 patients with inflammatory or neoplastic alterations were discussed. B cell or T cell type lymphocytes predominate with multiple sclerosis, T lymphocytes with tubercular meningitis. Varying quantities of B and T lymphocytes are found with viral meningitis. In one case the tumor cells of a reticulum cell sarcoma were identified in the CSF as T cells; in one case of plasmacytoma, tumor cells in the CSF were identified as B lymphocytes. In selected cases of leukemic or carcinomatous infiltration of the meninges and of medulloblastoma, CSF cells did not react to treatment with immunological markers. Key words: CSF ceils - Lymphocytes - Mononuclear phagocytes - I m m u n o logical markers - Cytochemistry.
Zusammenfassung. Es wird fiber Untersuchungen an Zellen des Liquor cerebrospinalis berichtet, die durch Anwendung verschiedener zytologischer Methoden zus~itzliche Informationen fiber die Herkunft und somit fiber die Art von pathologischen Ver~inderungen in den Leptomeningen erlauben. Bei den Methoden handelt es sich um die Anwendung von immunologischen und zytochemischen Markern zur Unterscheidung und Identifizierung von B- und T-Lymphozyten, von Zellen der Monozyten-Makrophagen-Reihe sowie von *
This study was supported by the Deutsche Forschungsgemeinschaft Address for offprint requests: Priv.-Doz. Dr. M. Oehmichen, Institut fiir gerichtliche Medizin der Universit~it, N/igelestr. 5, D-7400 Tiibingen, Federal Republic of Germany
0340-5354/78/0218/0187/$ 02.00
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M. Oehmichen and H. Huber n e u t r o p h i l e n G r a n u l o z y t e n und deren Vorlhufer. U b e r die p r a k t i s c h e A n w e n d u n g d e r M e t h o d e n an Liquorzellen von 16 Patienten mit entzt~ndlich o d e r t u m o r 6 s verfinderten L i q u o r e s wird berichtet. Bei m u l t i p l e r Sklerose finden sich s o w o h l L y m p h o z y t e n des B- als auch des T-Zelltyps vermehrt, bei t u b e r k u l 6 s e r Meningitis nahezu ausschliel31ich L y m p h o z y t e n des T-Zelltyps und bei Virusmeningitis B- und T - L y m p h o z y t e n in unterschiedlicher Relation. R e t i k u l o s a r k o m z e l l e n im L i q u o r c e r e b r o s p i n a l i s k o n n t e n in einem Fall als TZellen identifiziert werden, whhrend im Falle eines P l a s m o z y t o m s Zellen im L i q u o r c e r e b r o s p i n a l i s mittels i m m u n o l o g i s c h e r M a r k e r als Zellen der B-Zellreihe e r k a n n t werden k o n n t e n . Demgegenfiber zeigten die Zellen im L i q u o r c e r e b r o s p i n a l i s in Einzelf~illen von Meningosis leuk~imica, Meningosis carc i n o m a t o s a u n d bei M e d u l l o b l a s t o m keine R e a k t i o n nach der B e h a n d l u n g mit den i m m u n o l o g i s c h e n M a r k e r n .
Introduction W i t h i n general c y t o d i a g n o s i s , d e m o n s t r a t i n g surface receptors on cells of the l y m p h o c y t e cell series in the p e r i p h e r a l b l o o d is b e c o m i n g m o r e a n d m o r e i m p o r t a n t [2, 3, 8]. Criteria for classifying cells o f m a l i g n a n t l y m p h o m a p a r t i c u l a r l y have been o b t a i n e d in this way [13, 14, 16]. Only a few similar studies are available for cells o f the c e r e b r o s p i n a l fluid ( C S F ) [20]. Since these cells are d i a g n o s t i c a l l y relevant [10], p r e l i m i n a r y results for the d e m o n s t r a t i o n o f certain surface r e c e p t o r s o f C S F cells b y routine diagnostic p r o c e d u r e s are described. A n a t t e m p t was m a d e to develop a simple, inexpensive test which could be e m p l o y e d in m o s t l a b o r a t o r i e s . In some cases, it is necessary to perfect the differentiation a n d use a d d i t i o n a l controls. These p r e l i m i n a r y results for the a p p l i c a t i o n o f the technics to C S F cells f r o m patients with i n f l a m m a t o r y a n d / o r t u m o r o u s diseases o f the C N S m u s t be s u p p l e m e n t e d by d a t a f r o m a larger g r o u p of patients. Review o f the literature a n d critical e v a l u a t i o n are published elsewhere [20].
Material and Methods Basically, three different technics were used to classify the cells: the immunofluorescence lechnic, the rosette technic, and cytochemical methods. The investigations were carried out as follows:
I. Imrnunofluorescence Technic Approximately 5 ml of fresh CSF was mixed with 5 ml of physiological saline solution. The suspension was centrifuged at 120 to 150g/min for 5min at room temperature. All of the solution was discarded except for 1 ml of the sediment. To the sediment, 0.5 ml fluorescein isothiocyanate labeled serum (FITC) was added. Depending on the type of serum, the mixture was incubated for various lengths of time and at different temperatures (see below). The serum was then washed twice by adding 5 ml of physiological saline solution each time and centrifuged at 120 to 150 g/min for 5 min at room temperature. Approximately 1 ml of sediment remained after the last supernatant had been discarded. The sediment was carefully mixed with medium
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199Tc (Serva Feinbiochemica, D-6900 Heidelberg, FRG), and the mixture was then settled according to the Sayk method [19, 27]. The specimen was covered with glycerol (pH 7.0--7.2) and observed by fluorescence microscopy (microscope: Fa. Zeiss; Light source: mercury high pressure lamp, HBO 200W; Exciting filter: BG 12/4; Suppression filter: 53/44). Sera: The sera were- obtained from Behringwerke, D-3550 Marburg-Lahn, FRG, and Biologische Arbeitsgemeinschaft G m b H , D-6302 Lich, FRG. Since CSF cells are extremely sensitive to heat, they were incubated at 37 ° C tbr only 30 min and at 4 ° C for 60 rain. Polyvalent antihuman gamma globulin ( A H G ) from goat a n d / o r from rabbit (incubated usually at 37 ° C for 30 rain) was used as a B cell marker. Aggregated gamma globulin (AGG), see also [5], incubated at 4°C for 60 min, was used as a control and for further differentiation. Additional inhibition experirfients in which nonlabeled sera were added were undertaken as a control. After washing once, IgG or IgM (1 m g / m l each, Fa. Nordic Immunological Labs., Tilburg, Netherlands) was added to 1 ml sediment. The sediment was washed after 30 a n d / o r 60 min of incubation. 2. Rosette Technic Two methods each of which demonstrated different receptors were used: a) Rosette formation following pretreatment of sheep erythrocytes with neuraminidase (EN) to demonstrate T lymphocytes. The erythrocytes were pretreated according to previously described methods [17]. For each milliliter of CSF, 0.1 ml of an EN suspension adjusted to 16,000 erythrocytes/ml was added. After brief mixing, this suspension was centrifuged at 180 to 2 0 0 g / m i n for 5 min. The supernatant was carefully siphoned off. One drop of 30% bovine serum albumin (Fa. Serva, Feinbiochemica, D-6900 Heidelberg, FRG), two drops of medium I99Tc, and one drop of 1% toluidin blue solution were added. The rosette-forming round cells were immediately counted in a Fuchs-Rosenthal counting chamber; they were counted again 12h later. b) After having treated sheep erythrocytes with antisheep erythrocyte antiserum (Behringwerke, D-3550 Marburg-Lahn, FRG), phagocytosis experiments were carried out to establish Fc receptors in cells of the monocyte-macrophage cell series. After washing once with amboceptor (diluted 1:500) for 30 min at 37 ° C, the erythrocytes were sensitized (EA; for details of method, see [9]). A 0.5% EA suspension was produced after three washings. One milliliter of this suspension was added for every milliliter of CSF. The mixture was incubated for 30 min at 37 ° C and then settled in a Sayk chamber. After sedimentation, the glass slide was washed in physiological saline, and the specimen was stained according to May-Grtinwald/Giemsa (= Pappenheim).
3. Cytochemical Methods To confirm the findings, a few cytochemical analyses were made. They provided additional criteria for classifying the cellular elements in a cell series; the reaction products also served as markers. For all analyses, CSF was first settled according to the Sayk method. The following enzymes were demonstrated by hematological methods: alpha naphthyl acetateesterase (ANAE, 12) and acid phosphatase (Aptase, 15) as markers for cells of the monocyte-macrophage series; naphthol AS-D chloroacetateesterase (NAS-DCLAE, 12) as well as ANAE and Aptase were used as markers for granulocytes a n d / o r precursors of the myeloid cell series.
Evaluation As was expected, it was difficult to differentiate the individual cell types in the Fuchs-Rusenthal chamber. The numerical data obtained here should be regarded as only a rough estimate. In
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Fig. 1. Some CSF round cells forming rosettes after addition of neuraminidase pretreated sheep erythrocytes indicating T cell type lymphocytes (× 500) Fig. 2. Mononuclear CSF cell with phagocytized antigen-antibodycomplex, i.e. sheep erythrocytes pretreated with antibody, as an indication of a mononuclear phagocyte (May-Grfinwald/ Giemsa stain, × 1200)
each case, the cells can, however, be satisfactorily differentiated after sedimentation regardless of whether the cells were differentiated unstained by phase contrast microscopy (for the immunofluorescence investigations) or after May-Grfinwald/Giemsa staining. One hundred cells of each cell type or 100 cells per specimen were counted. The following cells type could be differentiated: a) mononuclear phagocytes including monocytes, histocytes, and macrophages; b) round cells with, for the most part, could be considered immunocompetent cells (e.g., lymphocytes); c) neutrophilic granulocytes; d) atypical cells such as tumor cells. The cells which exhibited clear-cut intracytoplasmatic fluorescence were considered as positive in the sense of FITC-labeled cells. Only those lymphocytes which had at least three erythrocytes adhering to their surface were considered positive in the sense of a rosette-forming cell (Fig. 1). With demonstration of the Fc receptor, these mononuclear phagocytes which had incorporated at least one erythrocyte were considered positive (Fig. 2). The cells which clearly demonstrated reaction products were considered enzyme positive.
Results The results of the differentiation in 11 patients with i n f l a m m a t o r y diseases of the l e p t o m e n i n g e s are presented in Table 1. The increase of cells with surface characteristics of m o n o n u c l e a r phagocytes in s u b a r a c h n o i d a l h e m o r r h a g e (2 patients) is c o n t r a s t e d with the increase of r o u n d cells (B lymphocytes as well as T lymphocytes) in multiple sclerosis (3 patients). In the patients with multiple
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Table 2. Classification of cells from various tumors (% of counted tumor cells in CSF) Diseases
Reticulum cell sarcoma Plasmacytoma Acute myeloid leukemia Stomach cancer Medulloblastoma
Cases (No.)
12 13 14 15 16
Methods of investigation for classification of Round cells
Mononuclear phagocytes
Leukocytes
AHG AGG EN rosettes
EA ANAE phagocytosis
NASDCLAE
0 91 0 0 0
0 0 0 0 0
0 72 0 0 0
88 0 0 0 0
0 90 100 90 (+)
Aptase
100 80 60 (+)
0 0 85 0 0
sclerosis, it s h o u l d be n o t e d that o n l y a few o f the m o n o n u c l e a r p h a g o c y t e s were able to react with the E A complexes u n d e r the e x p e r i m e n t a l c o n d i t i o n s (see discussion). A n increase in B l y m p h o c y t e s c o u l d be ascertained in two o f the three cases o f viral meningitis. W e f o u n d a considerable increase of l y m p h a t i c cells with surface characteristics o f T l y m p h o c y t e s in b o t h cases o f t u b e r c u l a r meningitis. Table 2 shows the results for five patients with various neoplastic diseases. R o u n d cells with surface characteristics o f T l y m p h o c y t e s were d e m o n s t r a t e d in one case o f r e t i c u l u m cell s a r c o m a (Fig. 3 a) with dissemination o f t u m o r cells in the l e p t o m e n i n g e s [21]. The t u m o r cells were rosette f o r m i n g (Fig. 3b). Pred o m i n a n t l y r o u n d cells with the ability for b i n d i n g a g g r e g a t e d I g G a n d Ig d e t e r m i n a n t s were f o u n d in one patient with p l a s m a c y t o m a (Fig. 4). T u m o r cells with characteristics o f T o r B l y m p h o c y t e s as well as m a c r o p h a g e s were not f o u n d in the C S F o f the o t h e r patients examined. Cytochemically, the m y e l o i d p r e c u r s o r cells were d e m o n s t r a t e d in the C S F o f a patient with leukemic i n f i l t r a t i o n o f the meninges. U n d e r o u r e x p e r i m e n t a l conditions, these cells as well as the m a t u r e g r a n u l o c y t e s did not show a n y I g G receptors. The results for one case each o f m e t a s t a s i z i n g cancer o f the s t o m a c h a n d m e d u l l o b l a s t o m a are p r e s e n t e d in T a b l e 2; they represent a negative control.
Discussion Based on this investigation, the following conclusions can be drawn: 1. Since o n l y a few cases were investigated, the results p e r t a i n i n g to i n f l a m m a t o r y diseases o f the l e p t o m e n i n g e s m u s t be c o n f i r m e d with a larger g r o u p o f patients. T h e findings were, however, s u b s t a n t i a t e d by the d a t a presented in the available literature. A relatively larger p e r c e n t a g e o f E A reacting, A N A E positive, a n d A p t a s e positive m o n o n u c l e a r p h a g o c y t e s were f o u n d in nonspecific i n f l a m m a t i o n o f the
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Fig.3a and b. Atypical CSF cells in a case of reticulum cell sarcoma. (a) May-Grtinwald/ Giemsa stain, × 1080. (b) Rosette forming tumor cells after addition of neuraminidase pretreated sheep erythrocytes indicating the T lymphocyte origin (× 450)
leptomeninges, represented here by the cases of subarachnoid hemorrhage [34, 35]. B type cells [25, 27] together with the simultaneous increase in the percentage of EN rosette-forming cells (to the contrary, see [6]) were demonstrated with multiple sclerosis. The obviously low percentage of EA phagocytizing m o n o nuclear phagocytes may possibly have been influenced by inhibition induced by the increased amounts of I g G usually present at the same time in the CSF [1, 5, 11, 26]. The reduced percentage of A N A E positive mononuclear phagocytes already described by Olischer [22, 23] was confirmed. A mixed T and B cell population was found in viral meningitis [4, 6, 7]. With tubercular meningitis, however, the round cell population is composed primarily of EN rosette-forming lymphocytes. Goasquen and Sabouraud [6] were also able to substantiate this finding. They observed spontaneous rosette formation in all of the 1000 round cells counted.
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Fig.4a--c. Atypical CSF cells in a case of plasmacytoma. (a) May-Griinwald/Giemsa stain, x 1200. (b) Phase displacement of the same cell (compare e) which shows a distinct fluorescence stain after treatment with anti human gamma globulin indicating the B lymphocyte origin (x 500)
2. The typical characteristics were found in the t u m o r cells. Only in malignant l y m p h o m a (histomorphologically identified as immunoblastic sarcoma) did the cells form EN rosettes which were apparently composed exclusively of T cells [I 3, 14, 16]. The experiments in which t u m o r cells from the same CSF specimen were incubated with antigen-antibody complex coated with the complement (EAC) p r o v e d negative, as did the PAS reaction. In p l a s m a c y t o m a with meningeal involvement, atypical cells were found with the characteristics o f the B cell series [29]. The receptors for aggregated g a m m a globulin and, to some extent, also for intracytoplasmic Ig were most often d e m o n s t r a t e d on plasmacyte precursors, but frequently not in mature cells from this series. These findings indicate that the leptomeninges were infiltrated with i m m a t u r e plasmacytes. The usual cytochemical alterations were f o u n d in adenocarcinoma of the stomach (for positive evidence of A N A E , see also [22, 23]) as well as myeloid leukemia (for positive evidence of N A S - D C L A E , see also [24]). Under our experimental conditions, however, the surface receptors could not be demon-
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strated. Cells in m e d u l l o b l a s t o m a showed neither surface receptors n o r a clearcut positive enzyme activity.
References 1. Cohen, S., Bannister, R.: Immunoglobulin synthesis within the central nervous system in disseminated sclerosis. Lancet 19671, 366--367 2. Cohnen, G.: FunktionundOberfl~ichenmarkermenschlicherT-und B-Lymphocyten. Dtsch. med. Wschr. 99, 2241--2245 (1974) 3. Cohnen, G., Augener, W., Buka, A., Brittinger, G.: Rosette-forming lymphocytes in normals and patients with malignant lymphomas. Acta haemat. 51, 65--75 (1974) 4. Dayan, A. D., Stokes, M. T.: Rapid diagnosis of encephalitis by immunofluorescent examination of cerebrospinal-fluid cells. Lancet 1973 I, 1977 5. Dickler, H. B., Kunkel, H. G.: Interaction of aggregated-globulin with B lymphocytes. J. exp. Med. 136, 191--196 (1972) 6. Goasguen, J., Sabouraud, O.: Rosettes mouton sur lymphocytes de liquide c~phalorachidien. Nouve Press. Med. 3, 2266 (1974) 7. Guseo, A.: Immunoglobulin-containingcells in the cerebrospinal fluid. Neuropat. Pol. (Warszawa) 12, 281--284 (1974) 8. Huber, C., Michlmayr, G., Huber, H.: Immunologische Marker in der Differentialdiagnose lymphatischer Systemerkrankungen. Dtsch. Med. Wschr. 99, 2262--2268 (1974) 9. Huber, H., Fudenberg, H. H.: Receptor sites of human monocytes for IgG. Int. Arch. Allergy 34, 18--31 (1968) 10. Jellinger, K., Radaskiewicz, T., Slowik, F.: Primary malignant lymphomas of the central nervous system in man. In: Symposium on Malignant Lymphomas of the Nervous System, Vienna 1974. Acta neuropath. (Berl.), Suppl.VI, 95--102 (1975) 11. KolfiL O., Ross, A. T., Hermann, J. T.: Serum and cerebrospinal fluid immunoglobulins in multiple sclerosis. Neurology (Minn.) 20, 1052--1061 (1970) 12. Leder, L.-D.: Der Blutmonocyt. Berlin-Heidelberg-New York: Springer 1967 13. Lennert, K.: Morphology and classification of malignant lymphomas and so-called reticuloses. In: Symposium on Malignant Lymphomas of the NervousSystem, Vienna 1974. Acta neuropath. (Berl.), Suppl. VI, 1--16 (1975) 14. Lennert, K., Stein, H., Kaiserling, E.: Cytologicaland functional criteriafor the classification of malignant lymphoma. Brit. J. Cancer 31, Suppl. II, 29--59 (1975) 15. L6ffler, H., Berghoff, W.: Eine Methode zum Nachweis von saurer Phosphatase in Ausstrichen. Klin. Wschr. 40, 363--364 (1962) 16. Lukes, R. J., Collins, R. D.: New approaches to the classification of the lymphomata. Brit. J. Cancer 31, Suppl. II, 1--28 (1975) 17. Michlmayr, G., Huber, C., Fink, U., Falkensamer, M., Huber, H.: T-Lymphocyten in peripherem Blut und Lymphknoten bei lymphatischen Systemerkrankungen. Schweiz. med. Wschr. 104, 815 (1974) 18. Oehmichen, M.: Characterization of mononuclear phagocytes in human CSF using membrane markers. Acta Cytol. 20,548--552 (1976) 19. Oehmichen, M.: Cerebrospinal Fluid Cytology. Stuttgart: Georg Thieme 1976 20. Oehmichen, M.: Differenzierung mononukle~trer Zellen des Liquor cerebrospinalis mit immunologischen und zytochemischen Markern. In: Die Cerebrospinalfliissigkeit (H. G. Mertens und D. Dommasch, Hrsg.). Stuttgart: Thieme 1978 21. Oehmichen, M., G~irtner, H.-V., Knittel-Jung, U.: T cell type immunoblastic sarcoma diagnosed primarily by CSF cell membrane features. Klin. Wschr. 55, 37--40 (1977) 22. Olischer, R. M.: Die Zellreaktionen des Liquor cerebrospinalis. Ergebnisse liquorzytologischer Untersuchungen in der Klinik entziindlicherund Tumor-Erkrankungendes Zentralnervensystems. Habil.-Schrift: Rostock 1969
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23. Olischer, R. M.: Der Nachweis der unspezifischen Esterase in Liquorzellen. Eine cytodiagnostische Zusatzuntersuchung. Z. Neurol. 200, 61--69 (1971) 24. Peiffer, J., Oehmichen, M., Ben6hr, H.-C., Frommhold, W., Rtickle, H., Waller, H. D., Wilmans, W., Wilms, KI.: Meningosis leucaemica bei Chlorom mit sp/iter leuk~imischer Generalisation. Dtsch. med. Wschr. 99,242--245 (1974) 25. Sandberg-Wollheim, M.: Immunoglobulin synthesis in vitro by cerebrospinal fluid cells in patients with multiple sclerosis. Scand. J. Immun. 3,717--730 (1974) 26. Savory, J., Heintges, M. G.: Cerebrospinal fluid levels of IgG, IgA and IgM in neurologic diseases. Neurology (Minn.) 23, 953--958 (1973) 27. Sayk, J.: Cytologie der Cerebrospinalfliassigkeit. Jena: VEB G. Fischer 1960 28. Schlote, W., Roos, W.: Gibt es ein charakteristisches Liquorzellbild bei multipler Sklerose? Beitrag zur Bedeutung der grofSen pyroninophilen Rundzellen im Liquor cerebrospinalis. Nervenarzt 45, 576--587 (1974) 29. Spaar, F.-W., Argyrakis: f.)ber Myelom-Zellen im Liquor cerebrospinalis. Z. Neurol. 202, 229--240 (1972) Received September 19, 1977
J. Neurol. 218, 187--196 (1978)
Neurology © by Springer-Verlag 1978
Supplementary Cytodiagnostic Analyses of Mononuclear Cells of the Cerebrospinal Fluid Using Cytological Markers* M. Oehmichen 1 and H. Huber 2 l Institut ftir Hirnforschung der Universit/it Ttibingen (Direktor: Prof. Dr. J. Peiffer), Calwer Strage 3, D-7400 Tfibingen, Federal Republic of Germany 2Medizinische Klinik der Universit/it Innsbruck (Direktor: Prof. Dr. H. Braunsteiner), A-6020 Innsbruck, Austria
Summary. Various methods of CSF cell differentiation are discussed. These methods provide additional information regarding the origin and, therefore, the types of pathological alterations occurring in the leptomeninges. They include the application of immunological and eytochemical markers to differentiate and identify B and T lymphocytes as well as cells from the monocyte-macrophage series and precursor cells of polymorphnuclear leukocytes. Application of these methods to CSF cells and the differentiation of CSF cells from 16 patients with inflammatory or neoplastic alterations were discussed. B cell or T cell type lymphocytes predominate with multiple sclerosis, T lymphocytes with tubercular meningitis. Varying quantities of B and T lymphocytes are found with viral meningitis. In one case the tumor cells of a reticulum cell sarcoma were identified in the CSF as T cells; in one case of plasmacytoma, tumor cells in the CSF were identified as B lymphocytes. In selected cases of leukemic or carcinomatous infiltration of the meninges and of medulloblastoma, CSF cells did not react to treatment with immunological markers. Key words: CSF ceils - Lymphocytes - Mononuclear phagocytes - I m m u n o logical markers - Cytochemistry.
Zusammenfassung. Es wird fiber Untersuchungen an Zellen des Liquor cerebrospinalis berichtet, die durch Anwendung verschiedener zytologischer Methoden zus~itzliche Informationen fiber die Herkunft und somit fiber die Art von pathologischen Ver~inderungen in den Leptomeningen erlauben. Bei den Methoden handelt es sich um die Anwendung von immunologischen und zytochemischen Markern zur Unterscheidung und Identifizierung von B- und T-Lymphozyten, von Zellen der Monozyten-Makrophagen-Reihe sowie von *
This study was supported by the Deutsche Forschungsgemeinschaft Address for offprint requests: Priv.-Doz. Dr. M. Oehmichen, Institut fiir gerichtliche Medizin der Universit~it, N/igelestr. 5, D-7400 Tiibingen, Federal Republic of Germany
0340-5354/78/0218/0187/$ 02.00
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M. Oehmichen and H. Huber n e u t r o p h i l e n G r a n u l o z y t e n und deren Vorlhufer. U b e r die p r a k t i s c h e A n w e n d u n g d e r M e t h o d e n an Liquorzellen von 16 Patienten mit entzt~ndlich o d e r t u m o r 6 s verfinderten L i q u o r e s wird berichtet. Bei m u l t i p l e r Sklerose finden sich s o w o h l L y m p h o z y t e n des B- als auch des T-Zelltyps vermehrt, bei t u b e r k u l 6 s e r Meningitis nahezu ausschliel31ich L y m p h o z y t e n des T-Zelltyps und bei Virusmeningitis B- und T - L y m p h o z y t e n in unterschiedlicher Relation. R e t i k u l o s a r k o m z e l l e n im L i q u o r c e r e b r o s p i n a l i s k o n n t e n in einem Fall als TZellen identifiziert werden, whhrend im Falle eines P l a s m o z y t o m s Zellen im L i q u o r c e r e b r o s p i n a l i s mittels i m m u n o l o g i s c h e r M a r k e r als Zellen der B-Zellreihe e r k a n n t werden k o n n t e n . Demgegenfiber zeigten die Zellen im L i q u o r c e r e b r o s p i n a l i s in Einzelf~illen von Meningosis leuk~imica, Meningosis carc i n o m a t o s a u n d bei M e d u l l o b l a s t o m keine R e a k t i o n nach der B e h a n d l u n g mit den i m m u n o l o g i s c h e n M a r k e r n .
Introduction W i t h i n general c y t o d i a g n o s i s , d e m o n s t r a t i n g surface receptors on cells of the l y m p h o c y t e cell series in the p e r i p h e r a l b l o o d is b e c o m i n g m o r e a n d m o r e i m p o r t a n t [2, 3, 8]. Criteria for classifying cells o f m a l i g n a n t l y m p h o m a p a r t i c u l a r l y have been o b t a i n e d in this way [13, 14, 16]. Only a few similar studies are available for cells o f the c e r e b r o s p i n a l fluid ( C S F ) [20]. Since these cells are d i a g n o s t i c a l l y relevant [10], p r e l i m i n a r y results for the d e m o n s t r a t i o n o f certain surface r e c e p t o r s o f C S F cells b y routine diagnostic p r o c e d u r e s are described. A n a t t e m p t was m a d e to develop a simple, inexpensive test which could be e m p l o y e d in m o s t l a b o r a t o r i e s . In some cases, it is necessary to perfect the differentiation a n d use a d d i t i o n a l controls. These p r e l i m i n a r y results for the a p p l i c a t i o n o f the technics to C S F cells f r o m patients with i n f l a m m a t o r y a n d / o r t u m o r o u s diseases o f the C N S m u s t be s u p p l e m e n t e d by d a t a f r o m a larger g r o u p of patients. Review o f the literature a n d critical e v a l u a t i o n are published elsewhere [20].
Material and Methods Basically, three different technics were used to classify the cells: the immunofluorescence lechnic, the rosette technic, and cytochemical methods. The investigations were carried out as follows:
I. Imrnunofluorescence Technic Approximately 5 ml of fresh CSF was mixed with 5 ml of physiological saline solution. The suspension was centrifuged at 120 to 150g/min for 5min at room temperature. All of the solution was discarded except for 1 ml of the sediment. To the sediment, 0.5 ml fluorescein isothiocyanate labeled serum (FITC) was added. Depending on the type of serum, the mixture was incubated for various lengths of time and at different temperatures (see below). The serum was then washed twice by adding 5 ml of physiological saline solution each time and centrifuged at 120 to 150 g/min for 5 min at room temperature. Approximately 1 ml of sediment remained after the last supernatant had been discarded. The sediment was carefully mixed with medium
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199Tc (Serva Feinbiochemica, D-6900 Heidelberg, FRG), and the mixture was then settled according to the Sayk method [19, 27]. The specimen was covered with glycerol (pH 7.0--7.2) and observed by fluorescence microscopy (microscope: Fa. Zeiss; Light source: mercury high pressure lamp, HBO 200W; Exciting filter: BG 12/4; Suppression filter: 53/44). Sera: The sera were- obtained from Behringwerke, D-3550 Marburg-Lahn, FRG, and Biologische Arbeitsgemeinschaft G m b H , D-6302 Lich, FRG. Since CSF cells are extremely sensitive to heat, they were incubated at 37 ° C tbr only 30 min and at 4 ° C for 60 rain. Polyvalent antihuman gamma globulin ( A H G ) from goat a n d / o r from rabbit (incubated usually at 37 ° C for 30 rain) was used as a B cell marker. Aggregated gamma globulin (AGG), see also [5], incubated at 4°C for 60 min, was used as a control and for further differentiation. Additional inhibition experirfients in which nonlabeled sera were added were undertaken as a control. After washing once, IgG or IgM (1 m g / m l each, Fa. Nordic Immunological Labs., Tilburg, Netherlands) was added to 1 ml sediment. The sediment was washed after 30 a n d / o r 60 min of incubation. 2. Rosette Technic Two methods each of which demonstrated different receptors were used: a) Rosette formation following pretreatment of sheep erythrocytes with neuraminidase (EN) to demonstrate T lymphocytes. The erythrocytes were pretreated according to previously described methods [17]. For each milliliter of CSF, 0.1 ml of an EN suspension adjusted to 16,000 erythrocytes/ml was added. After brief mixing, this suspension was centrifuged at 180 to 2 0 0 g / m i n for 5 min. The supernatant was carefully siphoned off. One drop of 30% bovine serum albumin (Fa. Serva, Feinbiochemica, D-6900 Heidelberg, FRG), two drops of medium I99Tc, and one drop of 1% toluidin blue solution were added. The rosette-forming round cells were immediately counted in a Fuchs-Rosenthal counting chamber; they were counted again 12h later. b) After having treated sheep erythrocytes with antisheep erythrocyte antiserum (Behringwerke, D-3550 Marburg-Lahn, FRG), phagocytosis experiments were carried out to establish Fc receptors in cells of the monocyte-macrophage cell series. After washing once with amboceptor (diluted 1:500) for 30 min at 37 ° C, the erythrocytes were sensitized (EA; for details of method, see [9]). A 0.5% EA suspension was produced after three washings. One milliliter of this suspension was added for every milliliter of CSF. The mixture was incubated for 30 min at 37 ° C and then settled in a Sayk chamber. After sedimentation, the glass slide was washed in physiological saline, and the specimen was stained according to May-Grtinwald/Giemsa (= Pappenheim).
3. Cytochemical Methods To confirm the findings, a few cytochemical analyses were made. They provided additional criteria for classifying the cellular elements in a cell series; the reaction products also served as markers. For all analyses, CSF was first settled according to the Sayk method. The following enzymes were demonstrated by hematological methods: alpha naphthyl acetateesterase (ANAE, 12) and acid phosphatase (Aptase, 15) as markers for cells of the monocyte-macrophage series; naphthol AS-D chloroacetateesterase (NAS-DCLAE, 12) as well as ANAE and Aptase were used as markers for granulocytes a n d / o r precursors of the myeloid cell series.
Evaluation As was expected, it was difficult to differentiate the individual cell types in the Fuchs-Rusenthal chamber. The numerical data obtained here should be regarded as only a rough estimate. In
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Fig. 1. Some CSF round cells forming rosettes after addition of neuraminidase pretreated sheep erythrocytes indicating T cell type lymphocytes (× 500) Fig. 2. Mononuclear CSF cell with phagocytized antigen-antibodycomplex, i.e. sheep erythrocytes pretreated with antibody, as an indication of a mononuclear phagocyte (May-Grfinwald/ Giemsa stain, × 1200)
each case, the cells can, however, be satisfactorily differentiated after sedimentation regardless of whether the cells were differentiated unstained by phase contrast microscopy (for the immunofluorescence investigations) or after May-Grfinwald/Giemsa staining. One hundred cells of each cell type or 100 cells per specimen were counted. The following cells type could be differentiated: a) mononuclear phagocytes including monocytes, histocytes, and macrophages; b) round cells with, for the most part, could be considered immunocompetent cells (e.g., lymphocytes); c) neutrophilic granulocytes; d) atypical cells such as tumor cells. The cells which exhibited clear-cut intracytoplasmatic fluorescence were considered as positive in the sense of FITC-labeled cells. Only those lymphocytes which had at least three erythrocytes adhering to their surface were considered positive in the sense of a rosette-forming cell (Fig. 1). With demonstration of the Fc receptor, these mononuclear phagocytes which had incorporated at least one erythrocyte were considered positive (Fig. 2). The cells which clearly demonstrated reaction products were considered enzyme positive.
Results The results of the differentiation in 11 patients with i n f l a m m a t o r y diseases of the l e p t o m e n i n g e s are presented in Table 1. The increase of cells with surface characteristics of m o n o n u c l e a r phagocytes in s u b a r a c h n o i d a l h e m o r r h a g e (2 patients) is c o n t r a s t e d with the increase of r o u n d cells (B lymphocytes as well as T lymphocytes) in multiple sclerosis (3 patients). In the patients with multiple
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Table 2. Classification of cells from various tumors (% of counted tumor cells in CSF) Diseases
Reticulum cell sarcoma Plasmacytoma Acute myeloid leukemia Stomach cancer Medulloblastoma
Cases (No.)
12 13 14 15 16
Methods of investigation for classification of Round cells
Mononuclear phagocytes
Leukocytes
AHG AGG EN rosettes
EA ANAE phagocytosis
NASDCLAE
0 91 0 0 0
0 0 0 0 0
0 72 0 0 0
88 0 0 0 0
0 90 100 90 (+)
Aptase
100 80 60 (+)
0 0 85 0 0
sclerosis, it s h o u l d be n o t e d that o n l y a few o f the m o n o n u c l e a r p h a g o c y t e s were able to react with the E A complexes u n d e r the e x p e r i m e n t a l c o n d i t i o n s (see discussion). A n increase in B l y m p h o c y t e s c o u l d be ascertained in two o f the three cases o f viral meningitis. W e f o u n d a considerable increase of l y m p h a t i c cells with surface characteristics o f T l y m p h o c y t e s in b o t h cases o f t u b e r c u l a r meningitis. Table 2 shows the results for five patients with various neoplastic diseases. R o u n d cells with surface characteristics o f T l y m p h o c y t e s were d e m o n s t r a t e d in one case o f r e t i c u l u m cell s a r c o m a (Fig. 3 a) with dissemination o f t u m o r cells in the l e p t o m e n i n g e s [21]. The t u m o r cells were rosette f o r m i n g (Fig. 3b). Pred o m i n a n t l y r o u n d cells with the ability for b i n d i n g a g g r e g a t e d I g G a n d Ig d e t e r m i n a n t s were f o u n d in one patient with p l a s m a c y t o m a (Fig. 4). T u m o r cells with characteristics o f T o r B l y m p h o c y t e s as well as m a c r o p h a g e s were not f o u n d in the C S F o f the o t h e r patients examined. Cytochemically, the m y e l o i d p r e c u r s o r cells were d e m o n s t r a t e d in the C S F o f a patient with leukemic i n f i l t r a t i o n o f the meninges. U n d e r o u r e x p e r i m e n t a l conditions, these cells as well as the m a t u r e g r a n u l o c y t e s did not show a n y I g G receptors. The results for one case each o f m e t a s t a s i z i n g cancer o f the s t o m a c h a n d m e d u l l o b l a s t o m a are p r e s e n t e d in T a b l e 2; they represent a negative control.
Discussion Based on this investigation, the following conclusions can be drawn: 1. Since o n l y a few cases were investigated, the results p e r t a i n i n g to i n f l a m m a t o r y diseases o f the l e p t o m e n i n g e s m u s t be c o n f i r m e d with a larger g r o u p o f patients. T h e findings were, however, s u b s t a n t i a t e d by the d a t a presented in the available literature. A relatively larger p e r c e n t a g e o f E A reacting, A N A E positive, a n d A p t a s e positive m o n o n u c l e a r p h a g o c y t e s were f o u n d in nonspecific i n f l a m m a t i o n o f the
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Fig.3a and b. Atypical CSF cells in a case of reticulum cell sarcoma. (a) May-Grtinwald/ Giemsa stain, × 1080. (b) Rosette forming tumor cells after addition of neuraminidase pretreated sheep erythrocytes indicating the T lymphocyte origin (× 450)
leptomeninges, represented here by the cases of subarachnoid hemorrhage [34, 35]. B type cells [25, 27] together with the simultaneous increase in the percentage of EN rosette-forming cells (to the contrary, see [6]) were demonstrated with multiple sclerosis. The obviously low percentage of EA phagocytizing m o n o nuclear phagocytes may possibly have been influenced by inhibition induced by the increased amounts of I g G usually present at the same time in the CSF [1, 5, 11, 26]. The reduced percentage of A N A E positive mononuclear phagocytes already described by Olischer [22, 23] was confirmed. A mixed T and B cell population was found in viral meningitis [4, 6, 7]. With tubercular meningitis, however, the round cell population is composed primarily of EN rosette-forming lymphocytes. Goasquen and Sabouraud [6] were also able to substantiate this finding. They observed spontaneous rosette formation in all of the 1000 round cells counted.
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Fig.4a--c. Atypical CSF cells in a case of plasmacytoma. (a) May-Griinwald/Giemsa stain, x 1200. (b) Phase displacement of the same cell (compare e) which shows a distinct fluorescence stain after treatment with anti human gamma globulin indicating the B lymphocyte origin (x 500)
2. The typical characteristics were found in the t u m o r cells. Only in malignant l y m p h o m a (histomorphologically identified as immunoblastic sarcoma) did the cells form EN rosettes which were apparently composed exclusively of T cells [I 3, 14, 16]. The experiments in which t u m o r cells from the same CSF specimen were incubated with antigen-antibody complex coated with the complement (EAC) p r o v e d negative, as did the PAS reaction. In p l a s m a c y t o m a with meningeal involvement, atypical cells were found with the characteristics o f the B cell series [29]. The receptors for aggregated g a m m a globulin and, to some extent, also for intracytoplasmic Ig were most often d e m o n s t r a t e d on plasmacyte precursors, but frequently not in mature cells from this series. These findings indicate that the leptomeninges were infiltrated with i m m a t u r e plasmacytes. The usual cytochemical alterations were f o u n d in adenocarcinoma of the stomach (for positive evidence of A N A E , see also [22, 23]) as well as myeloid leukemia (for positive evidence of N A S - D C L A E , see also [24]). Under our experimental conditions, however, the surface receptors could not be demon-
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strated. Cells in m e d u l l o b l a s t o m a showed neither surface receptors n o r a clearcut positive enzyme activity.
References 1. Cohen, S., Bannister, R.: Immunoglobulin synthesis within the central nervous system in disseminated sclerosis. Lancet 19671, 366--367 2. Cohnen, G.: FunktionundOberfl~ichenmarkermenschlicherT-und B-Lymphocyten. Dtsch. med. Wschr. 99, 2241--2245 (1974) 3. Cohnen, G., Augener, W., Buka, A., Brittinger, G.: Rosette-forming lymphocytes in normals and patients with malignant lymphomas. Acta haemat. 51, 65--75 (1974) 4. Dayan, A. D., Stokes, M. T.: Rapid diagnosis of encephalitis by immunofluorescent examination of cerebrospinal-fluid cells. Lancet 1973 I, 1977 5. Dickler, H. B., Kunkel, H. G.: Interaction of aggregated-globulin with B lymphocytes. J. exp. Med. 136, 191--196 (1972) 6. Goasguen, J., Sabouraud, O.: Rosettes mouton sur lymphocytes de liquide c~phalorachidien. Nouve Press. Med. 3, 2266 (1974) 7. Guseo, A.: Immunoglobulin-containingcells in the cerebrospinal fluid. Neuropat. Pol. (Warszawa) 12, 281--284 (1974) 8. Huber, C., Michlmayr, G., Huber, H.: Immunologische Marker in der Differentialdiagnose lymphatischer Systemerkrankungen. Dtsch. Med. Wschr. 99, 2262--2268 (1974) 9. Huber, H., Fudenberg, H. H.: Receptor sites of human monocytes for IgG. Int. Arch. Allergy 34, 18--31 (1968) 10. Jellinger, K., Radaskiewicz, T., Slowik, F.: Primary malignant lymphomas of the central nervous system in man. In: Symposium on Malignant Lymphomas of the Nervous System, Vienna 1974. Acta neuropath. (Berl.), Suppl.VI, 95--102 (1975) 11. KolfiL O., Ross, A. T., Hermann, J. T.: Serum and cerebrospinal fluid immunoglobulins in multiple sclerosis. Neurology (Minn.) 20, 1052--1061 (1970) 12. Leder, L.-D.: Der Blutmonocyt. Berlin-Heidelberg-New York: Springer 1967 13. Lennert, K.: Morphology and classification of malignant lymphomas and so-called reticuloses. In: Symposium on Malignant Lymphomas of the NervousSystem, Vienna 1974. Acta neuropath. (Berl.), Suppl. VI, 1--16 (1975) 14. Lennert, K., Stein, H., Kaiserling, E.: Cytologicaland functional criteriafor the classification of malignant lymphoma. Brit. J. Cancer 31, Suppl. II, 29--59 (1975) 15. L6ffler, H., Berghoff, W.: Eine Methode zum Nachweis von saurer Phosphatase in Ausstrichen. Klin. Wschr. 40, 363--364 (1962) 16. Lukes, R. J., Collins, R. D.: New approaches to the classification of the lymphomata. Brit. J. Cancer 31, Suppl. II, 1--28 (1975) 17. Michlmayr, G., Huber, C., Fink, U., Falkensamer, M., Huber, H.: T-Lymphocyten in peripherem Blut und Lymphknoten bei lymphatischen Systemerkrankungen. Schweiz. med. Wschr. 104, 815 (1974) 18. Oehmichen, M.: Characterization of mononuclear phagocytes in human CSF using membrane markers. Acta Cytol. 20,548--552 (1976) 19. Oehmichen, M.: Cerebrospinal Fluid Cytology. Stuttgart: Georg Thieme 1976 20. Oehmichen, M.: Differenzierung mononukle~trer Zellen des Liquor cerebrospinalis mit immunologischen und zytochemischen Markern. In: Die Cerebrospinalfliissigkeit (H. G. Mertens und D. Dommasch, Hrsg.). Stuttgart: Thieme 1978 21. Oehmichen, M., G~irtner, H.-V., Knittel-Jung, U.: T cell type immunoblastic sarcoma diagnosed primarily by CSF cell membrane features. Klin. Wschr. 55, 37--40 (1977) 22. Olischer, R. M.: Die Zellreaktionen des Liquor cerebrospinalis. Ergebnisse liquorzytologischer Untersuchungen in der Klinik entziindlicherund Tumor-Erkrankungendes Zentralnervensystems. Habil.-Schrift: Rostock 1969
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23. Olischer, R. M.: Der Nachweis der unspezifischen Esterase in Liquorzellen. Eine cytodiagnostische Zusatzuntersuchung. Z. Neurol. 200, 61--69 (1971) 24. Peiffer, J., Oehmichen, M., Ben6hr, H.-C., Frommhold, W., Rtickle, H., Waller, H. D., Wilmans, W., Wilms, KI.: Meningosis leucaemica bei Chlorom mit sp/iter leuk~imischer Generalisation. Dtsch. med. Wschr. 99,242--245 (1974) 25. Sandberg-Wollheim, M.: Immunoglobulin synthesis in vitro by cerebrospinal fluid cells in patients with multiple sclerosis. Scand. J. Immun. 3,717--730 (1974) 26. Savory, J., Heintges, M. G.: Cerebrospinal fluid levels of IgG, IgA and IgM in neurologic diseases. Neurology (Minn.) 23, 953--958 (1973) 27. Sayk, J.: Cytologie der Cerebrospinalfliassigkeit. Jena: VEB G. Fischer 1960 28. Schlote, W., Roos, W.: Gibt es ein charakteristisches Liquorzellbild bei multipler Sklerose? Beitrag zur Bedeutung der grofSen pyroninophilen Rundzellen im Liquor cerebrospinalis. Nervenarzt 45, 576--587 (1974) 29. Spaar, F.-W., Argyrakis: f.)ber Myelom-Zellen im Liquor cerebrospinalis. Z. Neurol. 202, 229--240 (1972) Received September 19, 1977
