CELLULAR METACHROMASIA I N CYSTIC FIBROSIS
G. B. REED, A. D. BAIN, W. M. MCCRAE AND F. M. SCOTT Department of Pathology, Royal Hospital for Sick Children, Universiiy of Edinburgh
CYSTIC fibrosis of the pancreas has been defined as an inherited disease in which there is a dysfunction of all or most of the exocrine glands (di Sant Agnese, 1964 ; Lobeck, 1966). The fundamental cause remains unknown and it has been difficult to suggest a pathogenesis recessively inherited yet leading to a general involvement of exocrine glands, especially when there is a disorder of ionic secretion in some glands and in others a disorder of seromucous secretion.
The demonstration of metachromasia in cultured fibroblasts from patients with this disease has directed interest to its study at a cellular level. Although the mechanisms by which this metachromasia is produced remain obscure, it has been reported to occur only when the fibroblasts were grown in media supplemented with newborn calf serum (Danes and Bearn, 1969). With this tissue-culture medium, no difference has been observed in the amount of metachromasia between those homozygous and those heterozygous for cystic fibrosis.
As cellular metachromasia has been observed in tissue cultures from normal individuals and in disorders other than cystic fibrosis the necessity for accurate definition of the in-vitro culture conditions must now assume considerable importance. There are apparently no reports of trials of fibroblast cultures from patients with cystic fibrosis in tissue-culture media supplemented with pooled human serum and relating the incidence of the metachromasia so obtained to the period in culture. Consequently the present investigation was undertaken to obtain information on this point and to see whether, by using pooled human serum, we could show any difference in the incidence of metachromasia between the homozygous patients and the heterozygous parents.
MATERIALS AND METHODS Skin biopsies from the forearm were set up as explant cultures in stoppered Pyrex baby
feeding bottles, with chicken plasma and thrombin, and gassed with 5 per cent. COz in air. The growth medium at this stage consisted of Eagles minimum essential medium (Burroughs Wellcome, based on Earles solution) together with chick embryo extract and 20 per cent. inactivated pooled human serum. Outgrowth on the average after 3-4 wk permitted sub- culture into further baby bottles and stock cultures were maintained in this way. Serial subcultures were taken at varying intervals into stoppered 12 x 150-mm test-tubes containing 2 ml of medium, 10 per cent. of serum and a flying coverslip (Paul, 1965). Coverslip pre- parations were examined after 24 hr and some at intervals of 4 and 6 days after subculture. Other subcultures were also made with medium containing human serum in varying con- centrations, with and without embryo extract and at intervals of 3-7 days. Before staining, the coverslips were washed twice in warm phosphate-buffered saline, fixed for 5 min. in methanol, air-dried and then stained with toluidine blue at pH 4.5 for 10 s (Culling, 1963). They were
Received 3 Dec. 1969; accepted 15 Feb. 1970. 8 . PATH.-VOL. 101 (1970) 251
then briefly rinsed in distilled water and mounted in DePex (Gurr). Each coverslip was examined in the same manner; ten fields with approximately 100 cells were examined along the long axis of the slide; alternate fields were chosen and the entire slide surveyed. All cells with metachromatic cytoplasm within the ten fields were counted, the total being expressed as the number of cells with metachromasia per thousand cells surveyed on each coverslip.
G. B. REED, A. D. BAIN, W. M. McRAE AND F. M. SCOTT
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FIG. 1.-Incidence of metachromatic cells in relation to time in culture (normal subjects).
RESULTS Our findings are based on cultures obtained from 9 normal children,
8 parents of children with cystic fibrosis and 11 children with cystic fibrosis ranging in age from 1 to 7 yr. Of the latter children, 6 were male and 5 female and all presented the characteristic triad of chronic pulmonary disease, pan- creatic deficiency and elevated sweat electrolytes. The time from primary explant to first subculture varied from 20 to 53 days, The number of subcultures
METACHROMASIA IN CYSTIC FIBROSIS 253
ranged from 5 to 25 and the total time in culture from 52 to 108 days. The morphological changes in the cells consist of cytoplasmic metachromasia similar to that previously described (Danes and Bearn, 1969). These authors separated the staining pattern of their cultures into two metachromatic cate- gories: type I , vesicular metachromasia (A with few vesicles, B with many vesicles) and type 2, diffuse vesicular and granular metachromasia. In our series type-3 cases are those without metachromasia.
In the present series of cultures from 19 individuals an attempt was made to follow this classification and, on a broad basis, 4 were classified as type 1, 12 as type 2 and 3 as type 3. Difficulties arose in this classification, however, as there was some overlap between type-I and type-2 metachromasia, depending on the coverslip field examined and the passages selected. Nevertheless, as
L L I I I I I I I I 1 1 I I I I ' I I I I I l ~
5 10 15 20 25 30 35 10 15 50 55 60 65 70 75 80 85 90 95 100 105 110
Days in culture FIG. 3.-hcidence of metachromatic cells in relation to time in culture (parents of subjects with
already noted by Danes and Bearn (1969), cultures from individuals in the same family appeared to be of the same classified type.
In the cultures from normal children, the percentage of cells showing metachromasia ranged from 0 to 2 per cent. (average 0.2 per cent.). In the cultures from children with cystic fibrosis, the metachromasia ranged from 0 to 19.3 per cent. (average 2.2 per cent.)-a ten-fold difference in the mean, This does not, however, reflect the actual distribution, which is better illustrated by the scattergrams (figs. 1 and 2) which, although emphasising the difference between normal individuals and patients with cystic fibrosis, show a wide variation in the amount of metachromasia observed in the latter. The amount of cellular metachromasia observed in a group of parents of children with cystic fibrosis (fig. 3) was similar to that observed in normal children. Variations in the amount of metachromasia were also found in the consecutive subcultures from the same patients with cystic fibrosis and it is of interest that on two occasions the amount of metachromasia in the particular case illustrated in fig. 4 was 0 and near 0. This is in contrast with the observations of Danes and Bearn (1968), who noted a progressive rise with subculture in the percentage of metachromasia to a constant 50-100 per cent.
J . PATH.-VOL. 101 (1970) R
254 G. B. REED, A. D. BAIN, W. M. McRAE AND F. M. SCOTT
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DISCUSSION Using reinforced Eagle's medium with 10 per cent. newborn calf serum,
Danes and Bearn (1968) have demonstrated that 50-100 per cent. of fibroblasts cultured from the skin in cases of cystic fibrosis exhibit cellular metachromasia. Cellular metachromasia has also been described in fibroblast cultures taken
- 1201 110
t 1 I I 1
1 I I I I I I I I I I
I I I I I I l
II u a '
10 20 30 40 50 60 70 80 90 100 1 I I I I 1 ,
Days in culture
FIG. 4.-Variation in incidence of metachromatic cells (one subject with cystic fibrosis).
from the skin in many other diseases (Matalon and Dorfmann, 1969). Cart- Wright, Danks and Jack (1969), however, have stressed the need for further study of the effect of growth rate and of subculturing on cellular metachromasia. Nadler et al. (1969) have also indicated that many conditions of tissue culture, including length of time in culture, relation of staining to subculture and composition of medium affect the presence and degree of metachromasia. Attention has also been drawn to the importance of the medium, and partic- ularly of its vitamin-C content (Punnett, Kistenmacher and Niederer, 1968). These observations appear relevant to the results obtained when the amount of cellular metachromasia in cystic fibrosis was plotted against the time of subculture.
Nevertheless, our results confirm that there is a difference between the
METACHROMASIA IN CYSTIC FIBROSIS 255
amount of metachromasia in cultures from patients with cystic fibrosis and cultures from normal children. The amount of metachromasia, however, was generally less than one-tenth of that previously reported (Danes and Bearn, 1968). The reasons for this are not yet apparent, but are probably related to the tissue-culture systems employed. The metachromasia, moreover, in our experience, varied in amount, not only