Nuclear Fluorescing Particles (F-Bodies) in Lymphocytes in Chronic Lymphocytic Leukaemia

Scand J Huemutol(1983) 3 0 , 3 7 4 3 7 8

Nuclear Fluorescing Particles (F-Bodies) in Lymphocytes in Chronic Lymphocytic Leukaemia IDA M. LISSE,NIELSH. HOLLANDER & J0RGEN L. THOMSEN Department of Pathology and Division of Haematology, Departmenr of Medicine, Hvidovre Hospital, Universiry of Copenhagen, Hvidovre, Denmark T h e number of fluorescing particles (F-bodies) in lymphocytes in 28 female and 45 male patients with CLL was compared to 63 (36 female, 27 male) controls, with and 25 (13 female, 12 male) without other haematological diseases. We found statistically highly significant increased F-body counts in the CLL groups, The present investigation indicates that the F-body feature described is specific for CLL-patients and may be a characteristic of the malignant clone of lymphocytes.

Key words: chronic lymphocytic leukaemia - F-bodies - nuclear fluorescence Accepted for publication December 14, 1982 Correspondence to: Dr. Ida M. Lisse, Department of Pathology, Hvidovre Hospital, DK-2650 Hvidovre, Denmark

The incidental finding of fluorescing particles (F-bodies) resembling Y-chromosomes in mononuclear (MN) cells in blood from females with chronic lymphocytic leukaemia (CLL) was described in a preliminary communication (Lisse et a1 1980). 3 out of 4 females with CLL had an F-body frequency in their MN cells clearly above the discrimination limit between males and females of 1 0 % . I n addition, an increased number of F-bodies were seen in males with CLL . This observation raised several questions regarding the diagnostic implications and the possible pathogenetic r61e of F-bodies in CLL. The present article describes the frequency of F-bodies in MN-cells from a larger series of patients with CLL as com-

pared to controls without and with haematological diseases other than CLL. In addition, we have examined isolated T- and B-lymphocyte populations from blood donors in order to elucidate whether extra F-bodies are a feature of normal B- or Tlymphocytes.

M A T E R I A L A N D METHODS

All patients with CLL seen in the Division of Haematology, Hvidovre Hospital, during the period of Apr 1980 t o Oct 1981 were included in the study. A total of 28 females (mean age 71.3, range 5C-87 years) and 45 males (mean age 70.5, range 52-82 years) were examined. 13 female patients (mean age 75.0, range 67-86 years) and 12 male patients (mean age 75.5, range

375

FLUORESCING BODIES IN CLL

fixed in Carnoy's solution for a minimum of 30 min and stained in quinacrine dihydrochloride (Thomsen 1975). Slides were examined in fluorescing light in blind trials. In each slide, MN and polymorphonuclear (PMN) cells were examined separately. 50 cells of each type were detected for the presence of F-bodies when possible. If less than 10 cells were found, the examination was regarded as non-conclusive. It should be emphasized that with regard to sex determination, the discrimination limit of 10% means 5 cells with one or more F-bodies out of 50 cells. The F-body index was defined as the total number of F-bodies per 50 cells.

66-89 years) without haematological disease were studied as a control group. Furthermore, 10 patients with acute myelocytic leukaemia (AML) (d = 5 , 9 = 5 ) , 5 patients with chronic myelocytic leukaemia (CML) ( d = 1, 0 = 4), 15 patients with myelomatosis, 3 patients with Waldenstrom's macroglobulinaemia and 1 patient with benign monoclonal gammopathy ( d = 9, 0 = lo), 3 patients with myelofibrosis ( d = 2, 9 = l), 19 patients with malignant lymphoma ( d = 8, P = 11) and 7 patients with other haematological diseases (acute lymphocytic leukaemia, benign lymphocytosis, plasmacytosis, unspecific haematological changes) (6 = 2, P = 5) were examined. In order to evaluate the distribution of F-bodies in normal T- and B-lymphocytes, we separated them from 3 blood donors in the following way: MN cells from peripheral blood were prepared by Lymphoprep@density centrifugation and separated in T-enriched and non-T-enriched subpopulations by rosetting with AETtreated sheep red blood cells and renewed Lymphoprep@density centrifugation.

Statistics. The Mann-Whitney rank sum test was used.

RESULTS

Figures 1 and 2 show the results of F-body detection in mononuclear cells in blood smears from females and males, respectively. It is seen that the mean F-body index is

F-body index. Blood with anticoagulant (EDTA) and the separated lymphocytes smeared on slides, air dried,

9

F- bodies per 50 MN

"T 25.

= Non-Hodgkin N=7

malignant lyrnfoma

A

= Myelomatosis N=10

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UN

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Mean age in years: Mean Noof F- bodies:

CLL

CONTROL

OTHER HEM.

71.3

75.0

6 8.9

9.3

0.5

2.0

No of pts.:

28

13

36

Figure 1. The occurrence of F-bodies per 50 mononuclears (F-body index) in females with CLL, without haematological diseases and with haematological diseases others than CLL. The mean value for CLL is indicated. See text for further information on the 2 patients with high F-body counts in the group of other haematological diseases.

376

L.ISSE, HOLLANDER & THOMSEN

higher in patients with CLL as compared to both patients without haematological diseases and to patients with haematological diseases other than CLL. 15 out of 28 females with CLL had an F-body index above 5 . We found a statistically significant difference (P < 0.001) between the F-body indices in females with CLL (mean 9.3, range 0-22), and females without haematological diseases (mean 0.5, range 0-2). The difference between the CLL females and the females with haematological diseases other than CLL (mean 2.0, range 0-17) was also highly significant (P < 0.001). None of the 13 female control patients and only 3 out of 36 female patients with haematological diseases other than CLL had an F-body index above 5. The patient with 6 F-bodies per 50 MN-cells had CML.

4

25 *

0

Figure 2. The occurrence of F-bodies per 50 mononuclears (F-body index) in males with CLL, without haematological diseases and with haematological diseases others than CLL. The mean value in each group is indicated.

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A

75 *

50

The 2 patients with 17 F-bodies had lymphoproliferative disorders with some CLLcharacteristics. One had a non-Hodgkin follicular malignant lymphoma in the spleen. The WBC reached 46.4 x lo9/] after splenectomy. Only 1 5 % of these were T-lymphocytes. 75 % were B-lymphocytes. The patient had low immunoglobulins (IgG = 5.2 g/l, 7.115.0; IgA = 0.17 g/l, 1.14-4.70; IgM = 0.29 g/l, 0.37-1.31). There were 40% lymphocytes in the bone marrow. The other patient had an initial WBC of 15.0 x 109/1 with 77% lympho-plasmocytoid cells in the peripheral blood and 86 % of the same cell type in the bone marrow. The diagnosis of myelomatosis was considered on the basis of bone lesions in the skull, free lambda-chains in the blood, 5 % plasma cells in the bone marrow and de-

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CONTROL OTHER HEM.

Mean aqe in years: Mean No of F- bodies:

70.5

75.5

64.8

41.9

22.5

2 3.6

NOof pts :

45

12

27

377

FLUORESCING BODIES IN CLL TABLE 1 Peripheral blood mononuclear cells from 2 normal females and I normal male

fluorescing particle in 35 patients (78%) compared to 2 out of 12 controls (17 %). The F-body indices in isolated T- and Blymphocytes from 3 blood donors are depicted in Table 1. There were no false positives (females with F-body indices > 10%) in PMN. There were, however, 25 out of 84 (30%) false negative (males with F-body indices S 10 %) in polymorphonuclears, and another 9 out of 84 non-conclusive (less than 10 PMN were found).

DISCUSSION

crease in renal function with an S-creatinine of 311 pmol/l (49-121). There were no statistically significant difference in F-body indices between females without haematological disease and females with haematological disease other than CLL (P = 0.5), but a significant difference (P < 0.001) between the number of F-bodies in male patients with CLL (mean 41.9, range 21-77) and the 2 control groups. There was no difference (P = 0.6) between the group of males without haematological disease (mean 22.5, range 15-35) and the male patients with haematological disease other than CLL (mean 23.9, range 0-46). The F-body indices in blood smears from males with CLL showed more than one

The occurrence of F-bodies exceeding the 10 % level is rare in normal females (Kringsholm et a1 1977). Two reports on ‘extra’ F-bodies in CLL have been published. Chrz et a1 (1972) reported this phenomenon in blood from a single female patient with CLL. Their primary interest seemed to be the consequences of this finding for the use of the method in sex determination; the possible association with a haematological disease was not investigated. LamborotManzur et a1 (1972) also reported on this phenomenon. Our initial observation of 4 female CLL-patients (Lisse et a1 1980) suggested that an abnormal F-body count may be a characteristic feature of this disease, supported by the present study where 15 out of 28 female CLL-patients showed this characteristic. In our opinion, the 2 female patients described above with high F-body counts may very well have an atypical CLL, or 2 malignant diseases. Various manifestations of one disease can not be excluded. The coincidence of myelomatosis and CLL has so far been described in 17 cases (Jeha et a1 1981).

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LISSE, HOLLANDER & THOMSEN

The present investigation thus indicates that the F-body feature described is highly specific for CLL. Since F-body determination is a simple technique, the method may have practical value for the diagnosis of CLL and in the differential diagnosis of B-lymphocyte diseases. In the present study, 37.5% of the female patients did not have an abnormal F-body count, and thus the sensitivity is rather low. The lack of false positives in PMN in CLL females suggests that the phenomenon described occurs exclusively for the lymphocytes. It is, however, difficult to make direct comparison between PMN and MN as Y-chromosome detection in PMN is difficult (Thomsen 1980). In Caucasians, more than 90 % of CLLcases are known to express B-lymphocyte characteristics. The results of the B- and T-lymphocyte studies indicate the possibility that the ‘extra’ F-body is a characteristic of the malignant clone of lymphocytes rather than an expression of numerical changes in the normal B- and T-lymphocyte populations. So far, we have not been successful in stimulation attempts on B-lymphocytes. Blymphocyte mitosis from CLL patients might reveal chromosomal abnormalities explaining the present finding. Gahrton et al (1980) reported on the successful stimulation on B-lymphocytes from CLL patients and found chromosome aberrations in 8 out of 11 patients. We have received blood smears from all of these patients. There was no correlation between the non-random chromosomal aberrations found by Gahrton et a1 (1980) and the results of our F-body

determination. The finding of ‘extra’ Fbodies, however, was also confirmed in the Swedish material (Gahrton et al 1980). The nature of the ‘extra’ F-body remains obscure.

ACKNOWLEDGEMENTS We are grateful to Dr. E. Dickmeiss, The Blood-Bank, Hvidovre Hospital and Dr. G . Gahrton and colleagues, Karolinska Institutet, Sweden for valuable help.

REFERENCES Chrz F, MichalovA K, Benesovii E & PossnerovA V (1972) A finding of F-body in blood cells of a woman with karyotype 46 XX with chronic lymphatic leukemia. Sb Lek 74,218-21. Gahrton G , RobCrt K-H, Friberg K, Zech L & Birg A G (1980) Non-random chromosomal aberrations in chronic lymphocytic leukemia revealed by polyclonal B-cell-mitogen stimulation. Blood 56, 6 4 0 4 7 . Jeha M T, Hamblin T J & Smith J L (1981) Coincident chronic lymphocytic leukemia and osteosclerotic multiple myeloma. Blood 57, 617-19. Kringsholm B, Thomsen J L & Henningsen K (1977) Fluorescent Y-chromosomes in hairs and blood stains. Forensic Sci Int 9, 117-26. Lamborot-Manzur M, Tischler P V & Atkins L (1972) Quantitative studies of Y-chromosomal fluorescence in human interphase nuclei. Clin Genet 3, 103-15. Lisse I M, Thomsen J L & Hollander N H (1980) Fluorescing particles (F-bodies) in lymphocytes in chronic lymphocytic leukaemia resembling Y-bodies. Scand J Haematol25,93-95. Thomsen J L (1975) Eine verbesserte Methode zum Nachweis des Y-chromosoms in Blutspuren. Z Rechrsmed 76, 81436. Thomsen J L (1980) An investigation of some of the factors influencing the results of Y-chromosome detection in blood stains. Forensic Sci Int 16. 111-17.