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General Guidelines and Principles

• Label slides, tubes, and slide holders: Use patient name and site. Labeling is necessary to prevent and correct sample errors, and to preserve chain of custody. A key, explained in the submission form, can be used for multiple masses/specimens.
• Submit premade smears: Submit premade unstained smears prepared immediately (<1 hour) after sample collection and quickly air dried. Cells degrade extremely quickly in fluids, and premade smears are necessary to preserve cellular morphology. Contaminating organisms may grow in transit, but premade smears can confirm in vivo sepsis.
• Send unstained smears: Stains are standardized in laboratories, so slight differences in staining of cellular components, infectious agents, and foreign materials are better identified with staining done at the laboratory. Some quick stains do not stain granules of mast cells, eosinophils and basophils.
• Send smears/samples with submission form in safe packaging: Complete the submission form as completely as possible. Keep slides dry and clean – do not refrigerate or expose to formalin fumes. Package well to prevent breakage.

What to submit

• Blood: Properly labeled EDTA blood and labeled unstained direct smears.
• Body Cavity fluids: Properly labeled EDTA fluid and labeled unstained direct smears. Sedimented smears are acceptable if identified as such. Collect and reserve a sterile red-top tube and/or culturette swab for potential aerobic and anaerobic culture or other additional tests (EDTA is bacteriostatic).
• Joint fluids: EDTA fluid for analysis and labeled unstained direct smears. Collect and refrigerate a culturette swab for potential aerobic and anaerobic culture (EDTA is bacteriostatic). Due to excessive sample thickness, sedimented slides are not useful.
• Washes: Unstained, properly labeled direct smears of fluid, sedimented smears, and smears of any floating particulate matter. A fluid analysis is not necessary. The typical blood smear technique with a very small droplet is best for direct and sedimented slides. Pull smears are best for floating particulate matter (transferred with a wooden stick). Note that cells degrade very quickly (15 minutes) because of the low protein content of wash fluid. Collect and reserve a sterile red top sample and/or culturette swab for potential aerobic and anaerobic culture, fungal culture, or PCR testing.
• Urine: Unstained, properly labeled, sedimented smears and a sterile red top tube of urine. Note that crystals degrade quickly in urine and cannot be seen on most cytology preparations.

Smear Techniques

Blood Smear Technique – Figure A (ideal with any fluid)

1. Use a needle tip or microhematocrit tube to place a very small drop on a clean slide near the white or frosted end of a properly labeled slide.
2. Place spreader slide above the drop away from the frosted edge, parallel to the slide below at a 30-45° angle.
3. Draw the spreader slide back into the drop, allowing the fluid to spread across the tip and make a line of fluid.
4. Quickly push the spreader slide away from the frosted edge to form a cone-shaped layer of blood/cells. The pointed end is the feathered edge.
5. Air dry quickly (not shown).

Practice Tips: Reduce droplet size if a feathered edge is not created. Change the angle of the spreader slide to adjust the length (a higher/larger angle shortens the smear while a smaller/lower angle lengthens it).

Pull Smear Technique – Figure B (do not use with blood)

1. Hold the slide with a small drop of fluid by the labeled white/frosted end in your non-dominant hand.
2. Place a clean slide, held in your dominant hand, over the fluid perpendicular (at 90°) to the bottom slide.
3. Lower the spreader slide onto the slide with fluid. When the two slides meet, the capillary action will hold the two slides together forming the shape of a cross.
4. While keeping the top slide perpendicular to the bottom slide, gently pull away from the frosted/white labeled end until the top slide is pulled completely off the bottom slide. When you are done, the slides will create the shape of a T (the bottom slide being the standing half and the top slide is the top half of the T).
5. Air dry quickly (not shown).Practice Tips: The capillary action causes significant cell trauma/disruption, and the pull smear technique is only best for thick samples such as particulate materials and transtracheal washes. Reduce droplet size if a feathered edge is not created.

Sediment Smears

Sedimented smears are made after centrifuging very dilute fluids such as body cavity effusions and transtracheal washes. Carefully decant all but about 0.5 ml of the supernatant fluid without disturbing the sediment. Gently mix the sedimented cells with the remaining fluid (do not shake) and make several smears of this sediment mixture using either the previously described Blood Smear or Pull Smear technique. Be sure to label sediment slides for accurate interpretation.