6.

Number of thymus-derived lymphocytes in normales and patients maintained on long-term kaemodialysis. Eckstein, D., D., U. Mey, R. Fuchs u. L. Lachhein: Folia Haematol., 106 (1979), 517 - 522

There is no doubt cell-mediated immunity is impaired by uraemia. The evidence for this includes prolonged graft survival (3,11)reduced delayed cutaneous responses to various antigens (3), (1, 16,20), abnormal in vitro lymphocytes responsiveness to different forms of stimulation (5, 14, 18), lymphocytes transfer test (2) and lyphopenia (20) However., the value of the indicated findings is not clear. This study was therefor designed to investigate if the number of thymus-derived lymphocytes in patients maintained on long therm haemodialysis is changed.

Materials and Methods

The population under investigation consisted of thirty seven patients with chronic renal failure maintained on long-term haemodialysis. The dialysis periods lasted for 6 or 7 h utilizing a Cordis Dow, Mocel 4. Ages of the pations ranged from 16 to 56 years with a mean of 35 years. There were thirteen woman and twenty four men. Serum creatinine ranged from predialysis values of 7,9 mg/100 ml to 15,8 mg/100 ml. Blood samples were collected prior to dialysis. The mean duration of haemodialysis before the study was 21 month with a range from 1 to 52 months. No patient was acutely ill when studied. Thymus-derived lymphocytes (T-rosettes) were identified by their capacity to form spontaneous rosettes with sheep red blood cells. Lymphocytes were seperated by density gradiation centrifugation ( first 20 min at 600 * g, second 10 min at 1500 * g). After washing there times in phosphate buffered saline (pH 7,4) the number of lymphocytes was adjusted (2,5 * 10\6/ml). A total of 0,25 ml of purified lymphocytes, 0,25 ml buffer and 0,125 ml of 5 % suspension of sheep red blood cells were mixed. Double tubes were set up for each patient. After centrifuging at 600*g additional incubation for 1 h (tube 1) and 24 h (tube 2) at +/- 4 °C was carried out. Before counting most of the supernatant was removed with a Pasteur pipette and the mixture was resuspended by gentle shaking. A rosette was defined as a lymphocyte with three or more adherent sheep red blood cells. The percentage of rosette-forming cells was determined by counting 200 lymphosytes. Peripheral blood leucocyte counts and lyphocyte counts were performed by standart methods. Total number of lymphocytes resulted by way of calculation. The absolute number of T-lymphocytes was then derived by multiplying the percentage of T-cells present and total lymphocyte counts. Vitality of lymphocytes was determined by using trypan blue. For the smear preparation the method deseribed by Fuchs et al. (6) was followed: After repeated gentle shaking the suspension of lymphocytes and sheep red blood cells was mixed with two drops of glutendialdehyde 0,5 % given by a Pasteur pipette . After smearing and drying 24 h staining was performed by standard methods (Fig. 1).

Results

Vitality
The mean value of dead and injured lymphocytes in 51 normals was 6,6 % (+/- 1 s.d. = +/- 5,1 %), in 37 patients, however, 6,2 % (+/- 1 s.d. = 4,6 %). There was no difference between the vitality of lymphocytes from normals and patients (Student`s t-test, P > 0,01).

Number of T-lymphocytes vs. Absolute lymphocytes count Incubating the suspension for 1 hr at 4 °C, the percentage of T-cells in the blood was 45,2 % +/- 11,8 % (mean +/- 1 s.d.) using native preparation and 42,7 % +/- 13,4 % (mean +/- 1 s.d.) using smear technique in normals compared with 40,4 % +/- 14,9 % (mean +/- 1 s.d.) using native preparation and 46,0 % +/- 10,0 % (mean +/- 1 s.d.) using smear technique in patients (Fig. 2). Incubation for 24 h at 4 °c showed 53,8 % +/- 16,3 % T-cells (mean +/- 1 s.d.) using native preparation and 51,5 % +/- 14,9 % (mean +/- 1 s.d.) using smear technique in normals, compared with 47,4 % +/- 12,2 % (mean +/- 1 s.d.) using native preparation and 48,1 % +/- 13,4 % (mean +/- 1 s.d.) using smear technique in haemodialysis pations (Fig. 3). There was no difference between normals and pations in forming T-rosettes (Student`s t-test of both native modifications, P > 0,01).

Table 1
Number of leucocytes, lymphocytes and T-lymphocytes per ml blood in patients maintained on long-term haemodialysis and in normals (mean +/- 1 s.d.)

	Leucocytes	Lymphocytes	T-lymphocytes (incubation time 1 h)	T-lymphocytes (incubation time 24 h)
Normals	5867 +/-2212 n=50	1769 +/- 614 n=50	790 +/- 297 n=48	946 +/- 470 n=43
Patients	6174 +/-2300 n=36	1681 +/- 675 n=36	617 +/- 365 n=32	789 +/- 408 n=29

Comparison of the test (native preparation) with 1 h and 24 h incubation in normals (P > 0,01) as well as in patients (P > 0,05) indicated slightly different values. The total number of leucocytes, lymphocytes (Tab. 1) in haemodialysis pations was not different from normals (P > 0,01 in all cases).

Diskussion

Identification of T-lymphocytes by their capacity to form spontaneous rosettes is today widespread and several modifications of the test have been reported. Some variations of the test performed in this work revealed two phenomena: The time of incubation at 4 °C (1 h and 24 h) has only a slight influence on the capacity of T-cells to form spontaneous rosettes. Seiler et al. (15) reported similar findings. Better in vitro stabilization of spontaneous rosettes of lymphocytes and sheep red blood cells using glutendialdehyde as reported by Fuchs et al. (6) for normals, can also be performed in chronically uraemic patients. In accordance with the study of Korz, Naber and Essers (8) the present findings show a normal vitality of uraemic lymphocytes. These results are in contrast to studies of Daniels et al. (4) and Reis et al. (13). Elves et al. (5),however, reported evidence of reducing the survival of normal leucocytes in uraemic serum and that centrifuged uraemic cell show "a better survival" in comparison to non-zentrifuged cells, maybe by destroying the moribund and less robust cells. Thus, there is the influence of uraemic serum in the findings of Daniels et al. (4) and Reis et al. (13) and, in our study, the effect of centrifugation which must be taken into consideration. That is why methodical aspects in these findings about vitality in lymphocytes from uraemic patients seem to be important. Lyphopenia in uraemia was reported by Wilson et al. (20) and Boulton-Jones et al. (1). In contrast, like Reis et al. (14), we found a normal number of lymphocytes in uraemia. It is also of interest if the number of T-lymphocytes is decreased. Sengar et al. (17). Reddy et al. (12) and Tong et al. (19) reported in this respect, but unfortunately there was no sufficient information on th number of lymphocytes in total and study populations were small. Other authors informed on normal total and relative T-lymphocytes in chronic uraemic patients (7,9,10,14). Our findings in a larger study populations show that the number of T-lymphocytes is normal. Thus, in conclusion, impaired immune responses of patients maintained on long-term haemodialysis is not due to lymphopenia and T-lymphopenia in blood. That is why further investigations into lymphocyte function and lymphocyte vitality in uraemia would be desirable.

Summary

For 32 patients in a chronic haemodialysis programme the number of thymus dependent lymphocytes was counted. These patients were susceptible to infections, the cause of which was supposed to be a disturbance of the immune defence. 51 healthy persons were taken as a control group. Various modifications of the spontaneous rosette test were applied. The number of T-lymphocytes proved to be as normal as the number of the total lymphocytes in all test series. This proves that the immune defect in patients with a chronic renal failure is not caused by T-lymphocytes as assumed by several authors.

References

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