Tseng’s modified method of specimen collection uses cellulose acetate filter paper from Millipore, which is trimmed into a 5 mm strip with one square end and one tapering end. The asymmetrical shape with a pointed tip facilitates grabbing and transferring the paper to the desired area with blunt smooth edged forceps.2 We use a 13 mm diameter Millipore paper divided in two "D"-shaped halves (fig 1). The end of the paper to be applied to the nasal side is clipped for orientation. One drop of local anaesthetic is instilled into the eye and excessive tear fluid and medication are wiped away. The paper is applied on the conjunctiva or cornea or both together, straddling the limbus. The area to be sampled depends on the underlying pathology. The filter paper is smoothed onto the ocular surface by applying gentle pressure with a Goldmann tonometer headpiece held between finger and thumb. The smooth flat surface of the headpiece allows uniform pressure to be applied over the surface area of the paper. The paper is allowed to remain in contact with the eye for approximately 510 seconds and then peeled off with a forceps. During the period of contact it is important that the lids are held away from the paper and it is not allowed to be wetted by tear fluid that may at times appear as a result of stimulation of lacrimation. If the paper gets unduly wet, the yield of cells will be poor. The paper is immediately transferred into a well of a 24 well plate containing fixative solution. It may be necessary to mark the back of the paper before applying it on to the ocular surface so that the surface to be stained later can be easily identified.

Figure 1  Schematic representation of impression cytology procedure as followed by the authors, showing cut filter paper disc into halves (left), and application of resulting D-shaped segments (clipped for orientation) on bulbar conjunctiva and cornea using Goldmann tonometer head, to apply even pressure on filter paper (right).

SPECIMEN STAINING

Papanicolaou or haematoxylin and PAS stains are the commonly used stains for routine histological staining of impression cytology specimens. The filter paper with the specimen is fixed for approximately 10 minutes in a solution containing glacial acetic acid, formaldehyde, and ethyl alcohol in a 1:1:20 volume ratio.2 A 24 well culture plate or a 24 well Teflon sample holder is used to hold the specimens during fixation and staining. The specimens are rehydrated in 70% ethyl alcohol and then placed successively in periodic acid Schiff reagent, sodium metasulfite, Gill’s haematoxylin, and Scott’s tap water substitute for 2 minutes each, rinsing in two changes of tap water in between each step.7 This is followed by dehydration in two changes of 95% ethyl alcohol, staining with modified orange G for 2 minutes, rinsing in 95% ethyl alcohol for 3 minutes, and staining with modified eosin Y for 2 minutes, again rinsing in 95% ethyl alcohol for 510 minutes, before dehydration in absolute alcohol for 5 minutes. Throughout the staining the cell side of the filter paper must be completely soaked with staining solution. For each destaining or rinsing, the holder is either dipped 10 times or suspended in a large jar with continuous magnetic stirring so that there is no need for constant monitoring.2 After the final destaining step, xylene is used to make the filter paper transparent. Before mounting, the filter paper is placed with the epithelial cells facing up. The completed slides are examined by light microscopy.

PREPARATION OF STAINING SOLUTIONS

Gill et al have described the detailed preparation of each solution.7 Gill’s haematoxylin is prepared by combining 365 ml of distilled water, 125 ml of ethylene glycol, 1 g of anhydrous haematoxylin, 0.1 g of sodium iodate, 8.8 g of aluminium sulphate, and 10 ml of glacial acetic acid. The chemicals are stirred for 1 hour on a magnetic mixer at room temperature. The final solution is filtered through Whatman No 1 filter paper before using it for the first time. Scott’s tap water substitute consists of 1 g sodium bicarbonate and 5 g magnesium sulfate, anhydrous or 10 g magnesium sulfate, crystalline in 500 ml of tap water. The pH of this solution is 8.02 and plays a significant part in determining the blue colour of the nuclei. Modified orange G is made of 10 ml orange G, 10% total dye content (TDC) aqueous solution combined with 490 ml of 95% ethyl alcohol and 0.075 g phosphotungstic acid. Modified eosin Y consists of 350 ml of 95% ethyl alcohol, 125 ml absolute methyl alcohol, 10 ml glacial acetic acid, 0.18 g light green SF yellowish, 5 ml 3% TDC aqueous solution, 10 ml eosin Y 20% TDC aqueous solution, and 1 g phosphotungstic acid.

SPECIAL TECHNIQUES

Special staining techniques have been devised for studying the specimens by electron microscopy3 and immunocytochemistry.4,5 For electron microscopy, the specimen on cellulose acetate paper is fixed in 4% phosphate buffered formaldehyde with 1% glutaraldehyde and ruthenium red dye, post fixed in buffered osmium fixative, dehydrated, and embedded in resin.3 Immunocytochemical staining of impression cytology specimens collected on conventional cellulose acetate membranes results in high levels of background staining. Use of xylene to chemically clear the cellulose acetate filter destroys the cell surface antigen. To overcome this problem Krenzer and Freddo developed a method of collecting the specimen on a pure nitrocellulose membrane which was then fixed with a spray fixative, transferred on to a poly-L-lysine coated glass slide, and dried. The slide was then placed in acetone for 1 hour with continuous agitation to dissolve the filter membrane, washed for 5 minutes in tap water, and subjected to cellulose digestion for 2 hours at 37°C to remove residual membrane material before proceeding to immunocytochemical staining.4 Thiel et al used mounted Biopore membranes (Millicell-CM 0.4 μm PICM 012550, Millipore Corp, Bedford, MA, USA) for the immunopathological diagnosis of superficial viral infections. The Biopore membrane is fully transparent in the wet state and allows a detailed cytological examination by light and fluorescence transmission microscopy.5

上一页  [1] [2] [3] [4] [5] [6] [7] [8] [9] [10]  ... 下一页  >>