Scand. /. ltntntinol.. Vol. 5, 1976.

Demonstration of T Lymphocytes in Cerebrospinal Fluid A. Department of Neurology and Broegelmann Research Laboratory for Microbiology, School of Medicine, University of Bergen, Bergen, Norway

Naess, A. Demonstration of T Lymphocytes in Cerebrospinal Fluid. Scand. ]. Immunol. 5, 165-168, 1976. Cerebrospinal fluid (CSF) was allowed to drop straight into Hanks's balanced salt solution. After centrifugation the pellet was resuspended and mixed with sheep erythrocytes. The mixture was further handled as in the E-rosette test with peripheral blood lymphocytes. CSF from 20 individuals were investigated, and rosette-forming cells (RFC) were found in all. Six patients with normal fluid had between 46% and 8 3 % RFC. Four patients with multiple sclerosis had increased numbers of RFC ( 9 4 % - 9 6 % ) . Low numbers of RFC were found in one patient with cerebellar ataxia and in one of two patients with acute viral meningitis. With this technique RFC can be counted even in normal CSF with a 3-ml sample. A. Neess, M.D., Medical Department, Diakonissehjemmets N-5000 Bergen, Norway

Rosette formation with untreated sheep erythrocytes (SRBC) has become an established method for the demonstration of T (thymusdependent) lymphocytes in peripheral blood (2, 5). This technique has also been applied for the demonstration of T lymphocytes in cerebrospinal fluid (CSF) (1, 6, 9). The number of lymphocytes in normal CSF is, however, low (less than 5 cells/mm^), and the methods available can only be used when the cell count is increased. Alternatively, one has to use rather large volumes of CSF. The present paper describes a simplified technique, which allows the counting of T lymphocytes in small samples of CSF, even if the total cell number is low.

sykehus,

Haraldsptass,

400 g for 40 min at room temperature and the supernatant removed. After the addition of 0.07 ml of 0.5% SRBC in HBSS, the mixture was incubated at 37°C for 5 min. The cells were centrifuged at 200 g for 5 min at room temperature and stored in ice water overnight. The supernatant was then removed. One drop of 0.6% glutaraldehyde in isotonic, phosphate-buffered saline, pH 7.2, temperature 4°C, was added to the cells (4). After incubation for 20 min the cells were resuspended and stained with a drop of methylene blue. One drop of the suspension was placed on a glass slide and covered with a cover slip. A minimum of 200 lymphocytes were counted. A rosette-forming cell (RFC) was defined as a mononuclear cell with lymphoid morphology MATERIALS AND METHODS binding more than three SRBC. Cerebrospinal fluid was obtained by lumbar Peripheral blood lymphocytes were obtained puncture. Three ml of CSF were allowed to by venipuncture. A modification of the techdrop from the cannula into a plastic tube con- nique of Jondal et al. (7) was used. The blood taining 6 ml of Hanks s balanced salt solution was immediately defibrinated with glass beads. (HBSS). The tube was then centrifuged at Two ml of defibrinated blood were mixed with

166

A. Ncess

Table I. Percentage of rosette-forming cells (RFC) in suspensions of peripheral blood lymphocytes counted before and after fixation with glutaraldehyde Patient no.

2 5 9 11 15 23 24 25 26 27

% RFC in suspension Unfixed

Fixed

60 51 69 60 52 56 66 50 31 77

60 54 65 59 54 59 65 52 33 73

Difference 0

-f3 -4 -1

+2 -f3 -1

Table II. Rosette-forming cells (RFC) in cerebrospinal fluid and peripheral blood from 20 patients Patient no. 1 2 3 4 5 6 7 8

+2 +2 -4

9 10

P > 0.1 (Wilcoxon test for two samples). 11

an equal part of physiological saline and carefully layered on 3 ml of Lymphoprep® (Nyegaard & Co,. Oslo, Norway) in a plastic tube. The tube was centrifuged for 40 min at room temperature, at exactly 400 g at the interface between blood/saline and Lytnphoprep (10). The cells from the interface were carefully pipetted off and washed three times in HBSS. The concentration was adjusted to 4 X 10^ cells/ml, and 0.25 ml of the lymphocyte suspension was mixed with 0.25 ml of 0.5% SRBC in HBSS. Incubation at 37°C, recentrifugation, and the remaining steps of the technique were performed as described above. As shown by others (8), fixation with glutaraldehyde did not influence the number of rosettes (Table I ) .

RESULTS Cerebrospinal fluid Rosette-forming cells were counted in CSF from 20 patients (Table II). Six patients (Nos. 1—6) had no neurological disorder that would cause CSF pathology, and their CSF cell counts and protein and IgG concentrations were normal. The proportion of RFC in the CSF of these patients was 4 6 % - 8 3 % . Four patients with acute exacerbations of multiple sclerosis had high ( 9 4 % - 9 6 % ) RFC counts in their

12 13 14 15 16 17 18 19 20

Diagnosis

%RFC

CSF Syncope Encephalopathy Encephalopathy Encephalopathy Encephalopathy Encephalopathy Encephalopathy Encephalopathy (and polymyalgia rheumatica?) Myelopathy Myelopathy and polyneuropathy Cerebellar ataxia (Marie's?) Disc prolapse Polyneuritis; porphyria( ?) Intraspinal myeloma Acute viral meningitis Acute viral meningitis Multiple sclerosis Multiple sclerosis Multiple sclerosis Multiple sclerosis

Blood

59 46 81 72 83 77 65 84

n.d.* 63 57 73 51 78 81 78

89 42

71 67

5

63

73 91

n.d. n.d.

68 32 57 96 94 95 96

77 52 n.d. 48 91 78 37

* n.d. = not done.

CSF. Low RFC counts in CSF were found in one patient with cerebellar ataxia and in one of two patients with acute viral meningitis.

Blood Rosette-forming cells were counted in peripheral blood from 16 of the 20 patients. Thirteen of these had RFC values within the normal range of this method, but, of the four multiple sclerosis patients, one had high ( 9 1 % ) and two had low ( 3 7 % and 4 8 % ) RFC counts in peripheral blood.

DISCUSSION The main obstacle to the study of RFC in CSF is the low number of lymphocytes. Of the three available reports, summarized in Table III, only

T Lymphocytes in CSF

167

Table III. Results of previous studies of rosette-forming cells (RFC) in cerebrospinal fluid (CSF) Authors

Goasguen & Sabouraud (6)

Allen & al. (1)

Sandberg-WoUheim & Turesson (9)

Diagnosis

No, of patients

Acute viral meningitis Tuberculous meningitis Multiple sclerosis Multiple sclerosis Stable Exacerbations 1st week 2nd week 3rd week Multiple sclerosis

4 1

3

% RFC Blood

CSF

0-15 10,5 9-25,6

30-80 100 0

29,6*

10

20

4

58-64

65* 38* 44,8* 70-83

* Mean values.

that of Goasguen & Sabouraud (6) describes the technique used in some detail. Samples of 20-25 ml of CSF were used, the cells were washed three times, SRBC added, and RFC counted immediately after centrifugation. This technique, then, dispenses with incubation. The other two reports are in the form of abstracts. Allen et al, (1) used the technique of Jondal et al, (7), The method of Sandberg-Wollheim & Turesson (9) could only be used for CSF with an increased cell count. The present technique is based on that of Jondal et al, (7), Some modifications are introduced. Gradient centrifugation and repeated washings have been dispensed with, thus minimizing cell loss. This has made it possible, in most cases, to obtain the necessary number of lymphoq'tes from only 3 ml of CSF, even in patients with normal CSF cell counts. Gradient centrifugation is used to separate lymphocytes from the other cells of peripheral blood. These cells are not found in normal CSF, however, and their presence—if any— in pathological fluid will be demonstrated by the staining of the cells before counting. Washing the lymphocytes has been found by some to increase the proportion of RFC in peripheral blood, and the presence of a serum factor inhibiting rosette formation has been

postulated (3). In the present study, more than 90% RFC has been found in the CSF of several patients, using lymphocytes unwashed but centrifuged through 6 ml of HBSS. The presence of any inhibiting factor in CSF is therefore regarded as highly unlikely. Thus the omission of gradient centrifugation and repeated washings should not affect the results. The percentage of RFC in normal cerebrospinal fluid was in the range of 46%-83%, Although this observation is based on CSF from only six patients, the data suggest that the normal range of CSF RFC found by this technique is similar to that of peripheral blood RFC, However, no evident correlation between blood and CSF values was found. Some patients had a higher proportion of RFC in blood than in GSF, others vice versa. The present results suggest that the proportion oif RFC in CSF may be almost 100% in acute exacerbations of multiple sclerosis (MS). This is a higher proportion than that found by Allen et al. (mean, 65%) (1) and Sandberg-Wollheim & Turesson (70%-83%) (9) (see Table III). In contrast, Goasguen & Sabouraud (6) could not demonstrate any RFC at all in the CSF of any of their MS patients (see Table III), The present technique therefore seems to be at least as sensitive as those previously reported.

168

A. Neess

ACKNOWLEDGEMENTS This investigation was supported by grants from Astri and Edvard Riisoens legat til fremme av vitenskapelig forskning, L, Meltzers Hoyskolefond, and Odd Fellows forskningsfond for multipel sklerose-sykdommen, REFERENCES 1, Allen, J,, Sheremata, W,, Cosgrove, J, B, R, & Osterland, K, Cerebrospinal fluid T and B lymphocytes kinetics related to exacerbations of multiple sclerosis. Neurology 2}, 352, 1975, 2, Bach, J. E, Evaluation of T-cells and thymic serum factors in man using the rosette technique. Trartsplant. Rev. 16, 196, 1973, 3 Chapel, H. M, The effects of papain, trypsin, and phospholipase A on rosette formation. Transplantation 15, 320, 1973, 4, Elliot, B, E, & Haskijl, J, S, Characterization of thymus-derived and bone marrow-derived rosette-forming lymphocytes, Europ. ]. Immunol. 3, 68, 1973,

Received 6 October 1975 Received in revised form 1 December 1975

5, Froland, S, S, & Natvig, J, B, Identification of three different human lymphocyte populations by surface markers. Transplant. Rev. 16, 114, 1973. 6, Goasguen, J, & Sabouraud, O, Rosettes mouton sur lymphocytes de liquide cephalo-rachidien. Nouv. Presse med. 3, 226, 1974. 7, Jondal, M,, Holm, G, & Wigzell, H. Surface markers on human T and B lymphocytes. I. A large population of lymphocytes forming nonimmune rosettes with sheep red blood cells. /. exp. Med. 136, 207, 1972. 8, Pang, G. T. M,, Baguley, D. M. & Wilson, J. D, Spontaneous rosetttes as a T-lymphocyte marker: a modified method giving consistent results. SRBC rosettes, /, immunol. Methods 4, 41, 1974. 9, Sandberg-Wollheim, M, & Turesson, I, B- and T-cells in the cerebrospinal fluid and peripheral blood in patients with multiple sclerosis. Proceedings of the XXI Congress of Scandinavian Neurologists, Stockholm, 1975. 10, Thorsby, E, & Bratlie, A, A rapid method for preparation of pure lymphocyte suspensions. In Terasaki, P. (ed.) Histocompatibility Testing. Munksgaard, Copenhagen, 1970.

Demonstration of T lymphocytes in cerebrospinal fluid.

Cerebrospinal fluid (CSF) was allowed to drop straight into Hanks's balanced salt solution. After centrifugation the pellet was resuspended and mixed ...
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