Photothermally induced vessel-wall necrosis after pulsed dye laser treatment: lack of response in port-wine stains with small sized or deeply located vessels.

The optimal treatment of port-wine stains is laser-induced selective photothermolysis. Lesion color and location and the age of the patient are reported to influence the therapeutic outcome. This study was initiated to analyze the outcome not only by the clinical response of lightening, but also in terms of photothermally induced necrosis to the vessel wall. Punch biopsy specimens were taken from 51 patients before treatment. Post-treatment biopsies were taken after exposure to a pulsed dye laser (585-nm wavelength, 0.45-ms pulse length) with an irradiant fluence of 6.5 J/cm2. Vessel diameter, depth, and wall thickness were measured in all histologic slides. The viability of the vessel walls was evaluated using an enzyme histochemical method. Port-wine stains with good blanching had significantly more superficially located vessels than the moderate and poor responders (p < 0.000). The moderate and good responding lesions consisted of moderate-sized vessels with diameters of 38 +/- 17 micrometers and 38 +/- 19 micrometers (mean +/- SD), respectively. The lesions showing poor blanching had significantly smaller vessels, with a diameter of 19 +/- 6.5 micrometers < 0.000). Analyses of the post-treatment specimens showed that coagulated vessels were superficially located and of moderate size, whereas the viable vessels were small with a median diameter of 14 micrometers. The probability of coagulation correlated with the thickness of the vessel wall. These data indicate that the therapeutic outcome of port-wine stains can be improved by using the lesional vessel parameters to select the optimal laser wavelength, pulse duration, and dose.

Laser treatment of port wine stains: therapeutic outcome in relation to morphological parameters.

Thirty patients were treated with a flashlamp-pumped pulsed dye laser, with 0.45 ms pulse width and 585 nm wavelength. Punch biopsies were taken prior to treatment, and the biopsies were examined morphometrically. Three different test sites were exposed to laser light of fluence 5.25, 6.50 and 7.75 J/cm2. The degree of blanching was examined 6-8 weeks after treatment, and each site was retreated four times. Six patients (20%) achieved poor blanching, eight patients (27%) obtained moderate lightening and 16 patients (53%) showed good response. The vessels of the good responders were located significantly more superficially than the vessels of the moderate and poor responders (P < 0.05). The poor responders had significantly smaller vessels than the moderate and good responders (P < 0.01). The moderate responders had deeper, but larger vessels, than the poor responders. Hence, an increasing vessel diameter reduces the negative outcome of increasing vessel depth. The vessel diameter was correlated to the colour (P < 0.01), e.g. the mean vessel diameter was increasing from 16.5 microns in pink lesions to 51.2 microns in purple lesions. The vessel depth was partly reflected in the lesional colour, as the pink and purple lesions had significantly deeper vessels than the red ones (P = 0.02). These results indicate that pink lesions predict poor blanching due to deeply located small vessels, while red lesions predict a good therapeutic result because of more superficially located vessels.

Immunoglobulin-containing cells in the colonic mucosa in patients with human intestinal spirochaetosis.

The relative proportions of cells containing the various classes of immunoglobulins in the colonic mucosa were determined in eight patients with human intestinal spirochaetosis (HIS) and in eight controls. All specimens were taken from colonic resections performed because of adenocarcinoma. None of the cases with HIS showed an inflammatory reaction in the mucosa as judged subjectively by light microscopy. Cases with HIS had smaller proportions of IgD- and IgE-positive cells and a larger proportion of IgA cells as compared with the controls, whereas the proportions of IgG and IgM cells were similar in the two groups. Taking into account the large individual variations in the proportions of immunoglobulins in both groups, our findings must be interpreted with caution. However, our results do not support the previous demonstration of increased percentages of IgE-positive cells in patients with HIS.

Cellular differentiation in prostatic explant cultures: assessed by electron microscopy and X-ray microanalysis.

A method developed for X-ray microanalysis (XRMA) of cell monolayers cultured on Formvar film has been shown to be well suited for the study of explant cultures of human prostate. Adherence and epithelial cell outgrowth occur as readily on the formvar as on ordinary tissue culture plastic, giving rise to cultures with the same morphological characteristics. Conventional transmission electron microscopy revealed intracytoplasmic granules with the appearance of secretory vesicles. XRMA of electron-dense intracytoplasmic granules in freeze-dried cryosections showed significant local sequestration of calcium, but not of magnesium or zinc, elements that have previously been shown to colocalize with calcium in secretory granules in vivo. It is concluded that some aspects of the secretory phenotype are supported in this in vitro model. The factors regulating the expression of a differentiated phenotype in prostatic epithelium await further elucidation. XRMA may be useful in assessing the effects on secretory differentiation induced by variations in the culture conditions.

Incorporation of contrast media in cultured cells.

Monolayer cultures of human prostatic (PC-3) and cervical (NHIK 3025) carcinoma cells were grown on formvar film and exposed to moderate concentrations of contrast agents for 30 minutes to 4 hours. After the exposure period, the monolayers were quickly frozen, and cryosections were examined by electron microscopy and X-ray microanalysis. Iodine was not detected in control cells, but was found in the cells that had been exposed to iodine-containing contrast media. The amount of intracellular iodine increased with increasing exposure dose and time. Because the cells mostly presented no sign of membrane damage, our findings support the view that contrast media have the ability to enter intact cells.

Rapid freezing of small biopsies and standard for cryosectioning and X-ray microanalysis.

A method for clamp-freezing of small biopsies for ultrathin cryosectioning is described. A formvar film is used to support the specimen before freezing with a pair of pliers, precooled in liquid nitrogen. This method makes it possible to align the piece of tissue close to a drop of standard solution and freeze the two simultaneously. After clamping, the flat, frozen sample contains both the piece of tissue and the standard in one solid block, which may thus be sectioned as a whole. The method has been used in the surgical theater, yielding good results with needle biopsies of the prostate and cystoscopically obtained biopsies of bladder tumors. The time lapse between excision and freezing may be less than 30 seconds, which represents a great improvement compared with the far more time-consuming mincing of tissue and mounting of the pieces on metal pins. From the frozen specimen, a large number of sections with good morphologic characteristics may be produced. The method was evaluated with the use of rat liver and lactating mammary tissue. Directly frozen tissue with intact blood supply was compared with biopsies prepared as described. Redistribution of Ca could not be demonstrated in either specimen type. However, the results indicate that the monovalent ions are far more mobile, and a significant increase of the Na:K ratios was observed in the biopsies compared with directly frozen tissue.

Intracellular distribution of calcium and zinc in normal, hyperplastic, and neoplastic human prostate: X-ray microanalysis of freeze-dried cryosections.

X-ray microanalysis has been performed on freeze-dried cryosections of normal, hyperplastic, and neoplastic human prostates. Needle biopsies from prostates suspected to be malignant were collected from ten patients. Seven of these patients had fully developed nodular hyperplasia, whereas the remaining three specimens were histologically classified as moderately differentiated adenocarcinomas. Prostates with normal light microscopic appearance were obtained from two brain-dead kidney donors. The concentration of calcium in secretory vesicles was found to be several orders of magnitude higher than the concentration of magnesium and zinc in all of the glands studied, which is consistent with calcium being the major cation secreted by the prostatic acinar cells. Some elderly prostates and neoplastic prostates revealed even lower mean zinc:calcium ratios in secretory vesicles. Even though both elements varied considerably, the wide variation of the zinc:calcium ratios was due mainly to variations in the concentration of zinc. Large, electron-dense bodies occasionally were found in the cytoplasm of prostatic acinar cells. These bodies contained high concentrations of sulphur which by far was the dominating element.

Quick sampling and perpendicular cryosectioning of cell monolayers for the X-ray microanalysis of diffusible elements.

A quick sampling and preparation method for freezing of cell monolayers is described. The cells are grown on a large Formvar film supported by a frame of polystyrene. A polyvinylpyrrolidone (PVP) solution is applied to one side of the film forming a flat disc when frozen with a pair of pliers precooled in liquid nitrogen. The PVP solution provides the specimen with sufficient strength and may be used as an elemental standard for absolute quantification if salts of known concentrations are added. Manipulation of the cells prior to freezing is thus restricted to a minimum, which eliminates possible harmful treatments like scraping and centrifugation. The procedure is quickly performed, the freezing being completed within 30 s of the cells having been removed from the culture well. The analytical results reveal low and stable Na:K ratios. Our results confirm that cells in vitro are comparable to cells in vivo with respect to elemental composition.

Subcellular concentrations of calcium, zinc, and magnesium in benign nodular hyperplasia of the human prostate: X-ray microanalysis of freeze-dried cryosections.

Biopsies from human prostates were obtained from normal and hyperplastic glands. The intracellular concentrations of calcium, zinc, and magnesium were analyzed using X-ray microanalysis of freeze-dried cryosections. Two prostate biopsies were obtained from kidney donors, ages 19 and 50 years, without any sign of benign nodular hyperplasia. The normal tissues were frozen within 15 min after circulatory arrest. The central part of biopsies from eight elderly men suffering from benign nodular hyperplasia were frozen within 30 s after excision. Adjacent tissue was processed for light microscopy and histopathological diagnosis. All samples were fresh-frozen using liquid nitrogen cooled pliers, without the use of any freeze-protection, fixation, or staining. In both the normal and the hyperplastic prostates high concentrations (up to above 100 mmol/kg dry weight) of zinc were present in electron dense bodies in the cytoplasm of the epithelial cells. Together with zinc, about equal concentrations of magnesium were found. Calcium was detected in 4 to 8 times the concentration of zinc. Significant, positive correlation between calcium and zinc as well as between calcium and magnesium in the cytoplasm was a typical finding in both normal and hyperplastic glands. In six of eight patients, older than 60 years of age, high levels of calcium (17.0-38.8 mmol/kg dry weight) were observed in the nuclei of the epithelial cells, while very low values were found in the remaining two. In the two younger cases (19 and 50 years of age), the nuclear calcium level in prostatic epithelium was relatively low (about 10 mmol/kg dry weight). These observations suggest that an increase of intranuclear calcium with advancing age may be of pathogenetic significance to growth disturbances in the prostate.

Idiopathic haemochromatosis and eye symptoms. A case report.

A 61-year-old man with idiopathic haemochromatosis and eye symptoms is described. The examination showed microaneurysms scattered over the bulbar conjunctiva, and some discrete pigmentations in the superficial tissue in the perilimbal area. Each cornea revealed a light brown, ellipsoid opacity with the longest diameter horizontally, seemingly being located in the corneal epithelium and/or the anterior part of the stroma. A corneal biopsy was examined by light and electron microscopy. Two different types of granules were found mainly located to the Bowman's membrane. X-ray microanalysis showed no detectable amounts of iron, but high concentrations of phosphorus and calcium, and in addition magnesium in one type of the granules. Treatment with EDTA improved the vision considerably.

A section press and low elemental support for enhanced preparation of freeze-dried cryosections.

A press- and specimen holder system is described, whereby thin, freeze-dried cryosections can be obtained more conveniently and with greater protection against contamination and harmful temperature fluctuations. The use of a grid and retainer assembly as the basic working unit, greatly facilitates low temperature work particularly when delicate grids are preferable from an analytical point of view. A new press has been constructed in order to keep the sections permanently pressed during freeze-drying and rewarming. The press also protects the sections during transfer to an external freeze-dryer.

Theoretical analysis of the ice crystal size distribution in frozen aqueous specimens.

To estimate theoretically how suited different freezing techniques are for freezing of freeze-etch specimens, it is necessary to know the relationship between specimen cooling rate and the resulting average ice crystal size. Using a somewhat simplified theoretical analysis, we have derived the approximate ice crystal size distribution of nonvitrified frozen aqueous specimens frozen at different cooling rates. The derived size distribution was used to calculate the relationship between relative change in average ice crystal size, (delta l/l), and relative change in specimen cooling rate delta (dT/dt)/(dT/dt). We found this relationship to be (delta l/l) = -k X delta (dT/dt)/(dT/dt) where k = 1.0 when specimen solidification takes place at about -6 degrees C, and k congruent to 1.3 when it takes place at about -40 degrees C.

Theoretical analysis of specimen cooling rate during impact freezing and liquid-jet freezing of freeze-etch specimens.

We have carried out a theoretical analysis of specimen cooling rate under ideal conditions during impact freezing and liquid-jet freezing. The analysis shows that use of liquid helium instead of liquid nitrogen as cooling medium during impact freezing results in an increase in a specimen cooling rate of no more than 30-40%. We have further shown that when both impact freezing and liquid-jet freezing are conducted at liquid nitrogen temperature, the two methods give approximately the same specimen cooling rate under ideal conditions except for a thin outer layer of the specimen. In this region impact freezing yields the highest cooling rate.