Gamma-glutamyltranspeptidase-positive hepatocyte foci were produced in female rats given a single dose of diethylnitrosamine neonatally after birth and, after weaning, a diet containing phenobarbitone for 30 wk. The nucleator method, a new stereological approach, provided an efficient, unbiased estimate of mean cell volume in focal lesions and extrafocal areas. It also provided an unbiased sample of cells to estimate hepatocyte nuclear volume and the percentage of binucleated cells. The results showed an increase in the mean volume of mononucleated cells--from 4,700 micron3 in extrafocal areas to 12,700 micron2 in foci--and of binucleated cells--from 6,900 micron3 to 25,000 micron3. This demonstrated the hypertrophic effect of the carcinogenic treatment in focal lesions. A striking reduction in the proportion of binucleated cells was also observed in the preneoplastic lesions. Nuclear volume measurements from mononucleated and binucleated hepatocytes were used to assess ploidy. An apparent increase in nuclear ploidy, with no change in cellular ploidy, was noted in focal tissue when compared with nonfocal tissue. This appeared to be caused by an increase in mononucleated tetraploid cells and a reduction in binucleated cells with two diploid nuclei, indicating an altered mitotic mechanism in focal lesions. The significance of these changes in cell volume, apparent ploidy levels and binuclearity in preneoplastic foci is discussed in relation to the hepatocarcinogenic process.

We report three cases of ring chromosome 5 [r(5)], two familial (mother and daughter) and one sporadic. The phenotype resembled that of the "ring syndrome" with prenatal onset of short stature, growth retardation, mild facial dysmorphism and normal psychomotor development. Extended metaphase and prometaphase chromosome preparations using G-, R- and Q-banding and scanning electron microscopy (SEM) failed to demonstrate deletion in the ring 5. Flow karyotype using the FACS cell sorter and peak area analysis showed the r(5) to be in the same position as the normal chromosome 5. The deletion that is presumably associated with ring formation appears to involve less that one megabase of DNA. In the "complex" rings, high resolution SEM showed fragile sites at the 5q34 and 5q35 region with frequent deletions at that site. A literature survey suggests that when a parent carries a ring chromosome about 80% of recognised pregnancies result in live birth. Of these, about half have a normal phenotype and karyotype, and half inherit the parental ring; about half of those acquiring the ring (20%) show significant mental retardation.

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SUMMARY Scanning electron microscopy (SEM) has been used to study the fragile X chromosome.The fragile site appears as an isochromatid gap in the majority of cases, confirming light microscope (LM) observations. SEM has allowed a more precise location of the fragile site to the Xq27-3 region.Light microscope (LM) studies have indicated that the fragile site on the X chromosome is most frequently observed as a non-staining gap of variable width, usually involving both chromatids. However, there is some uncertainty as to the exact position of this site. Sutherland1 2 defined it to be either in the Xq27 or Xq28 region. The enhanced resolution provided by scanning electron microscopy (SEM) has allowed both confirmation of the morphology and precise location of the fragile site by application of a technique used previously to karyotype human metaphases by SEM.3 Materials and methodsHeparinised peripheral blood, from a male patient known to have a fragile X chromosome, was cultured in TC 199 medium with Earle's Salts without serum supplementation and containing phytohaemagglutinin for 66 hours at 37°C. A total of 0 01 ,ug/ml colcemid was added for 1 hours. Cells were incubated in 75 mmol/l KCI for 5 minutes at room temperature followed by three washes in methanol-acetic acid fixative (3:1). Between the second and third washes cells were incubated for I hour at -20°C. Chromosomie spreads were made by dropping suspension onto coverslips, followed by air-drying. Several coverslips were stained in 20% Giemsa in Gurr's buffer (pH 6.8) for 8 minutes at room temperature for LM analysis.Two to three days later the remaining coverslips were G banded using a method modified from the technique of Seabright4 as follows: coverslips were incubated in 0.025% trypsin in 0.8504 saline for