Slide Preparation

Abstract

1691—Bonanni published first illustration of a “slider.”¹ Early 1800s—Slider transitions to slide.² 1840—Microscopical Society of London sets 3×1-in. as the standard size for micro slides.³ 1957—Frosted “Dakin” slide patented.4 1970—ASTM publishes its fi rst Standard Speci fi cation for Cover Glasses and Glass Slides for Use in Microscopy.⁵

“Adhesion flattens and retains cells; cohesion fattens and loses cells.”

Apppendix A. Saponin Technique for Bloody Fresh Cell Suspensions

When erythrocytes outnumber nucleated cells in specimens from any body site, they exclude such cells—including cancer cells—from micro preparations. These preparations are technically satisfactory but functionally unsatisfactory, meaning that the preparations represent the true mix of cells in the raw specimens and have been well prepared but are useless for cancer detection. Specifically the preparation method has invalidated the value of the raw specimen. The results are reported as within normal limits, when in fact the preparation method is unsatisfactory and the reported result is a false negative.

This common problem can be remedied by eliminating erythrocytes from cytologic specimens before the cell concentrate is collected on slides or filters. Such an approach to specimen enrichment is entirely different than hemolyzing RBCs after a cell spread is prepared by immersing it in a Carnoy’s-type hemolytic fixative or in 2 M urea. The latter techniques merely increase the visibility of the remaining cells but leave their numbers unchanged.

The saponin method that follows hemolyzes erythrocytes while in suspension, thus proportionally increasing the number of nucleated cells available for microscopic examination and permitting these cells to occupy the additional collection/display area that is now available. This enrichment technique was used in a research project on circulating cancer cells in peripheral blood.

The first time it was used for a clinical application, the control preparations exhibited countless erythrocytes but no cancer cells. The experimental preparations that had been processed with the saponin method described below, on the other hand, exhibited the exact opposite results: no erythrocytes and an abundance of cancer cells. If the specimen had not been processed with saponin, it would have been reported as negative and satisfactory. In other words, it would have been a laboratory technique-based false negative.

This technique should be applied to all cytologic specimens in which erythrocytes are visible in the cell concentrate—no matter how small—following the initial centrifugation.

A.1 Materials

• Hemolytic agent: 1% (w/v) saponin in distilled water with 0.2% sodium p-hydroxybenzoate as a preservative (optional). Filter through a 5 µm pore size cellulosic filter after preparation (also optional). Keep refrigerated

• Antihemolytic agent: 3% (w/v) calcium gluconate in distilled water with 0.2% sodium p-hydroxybenzoate (optional). Filter as above. Keep refrigerated

• Balanced electrolyte solution (not normal saline)

• 50-mL plastic centrifuge tubes

• Transfer pipettes

• Vortex mixer

A.2 Method

• 1. Centrifuge the specimen, up to 50 mL, for 10 min at 3,000 rpm.

• 2. Discard the supernatant.

• 3. Add 25 mL balanced electrolyte solution.

• 4. Resuspend the cell concentrate by repeatedly inverting the centrifuge tube, or better, by agitating the contents on a vortex mixer.

• 5. Add balanced electrolyte solution to the 45-mL level and mix.

• 6. Add 2 mL saponin and invert several times to mix.

• 7. After 1 min, add 3 mL calcium gluconate.

• 8. Centrifuge 10 min at 3,000 rpm.

• 9. Decant the supernatant.

• 10. Prepare cell spreads if volume permits. Otherwise, resuspend the button in 5 mL balanced salt solution for collection by cytocentrifugation or membrane filtration.

A.3 Results

After step 8, the supernatant will be colored red (the depth of color is a function of amount of hemoglobin released by the RBCs); the cell concentrate, white. RBC ghosts remain suspended in the supernatant and thus cannot contaminate the cell concentrate. The increased number of nucleated cells is remarkable. Cancer cells are often present in the specimen that otherwise would be absent in the final preparation.

A.4 Discussion

The value of the results is self-evident. The relative amount of RBC to nucleated cells determines whether the specimen should be treated with saponin. In other words, even small total numbers qualify for saponin enrichment. The saponin and calcium gluconate solutions may grow microbes. It is uncertain how long saponin solutions remain effective. Historically, we discarded saponin solutions after 1 week. Saponin is a plant derivative that varies in activity. Some lots may require higher concentrations and/or longer exposure times. Saponin hemolyzes RBCs by etching holes in the membranes, thus allowing hemoglobin to escape. Nucleated cells will begin to show cytoplasmic damage if exposed to saponin for too long. Saponin does not work in alcohol-preserved specimens.