Typically, fluid should be collected aseptically into an appropriately-sized tube containing EDTA. If fluid volume permits, fluid may be submitted in an EDTA tube and a red top clot tube (e.g. for potential culture). NOTE: Placement of a small amount of fluid into certain large EDTA-containing tubes will falsely increase specific gravity and total protein concentration, and may cause enough osmotic cell shrinkage to interfere with cytologic evaluation. A tube without anticoagulant may be used for samples that are not expected to clot (e.g. those without blood and without marked protein exudation).

Because cells deteriorate rapidly in many fluid samples, please prepare both direct smears and concentrated smears as soon as possible following collection to submit with the fluid. Be sure to label the smears as direct or concentrated. Additional tips for fluid cytology handling and slide preparation can be found at in two previous newsletter articles: Fluid Cytology Preparations and Frequently Asked Questions in Clinical Pathology.

To prepare concentrated smears:

• Centrifuge an aliquot of the fluid sample.
• Remove a portion of the supernatant.
• Resuspend the cellular material.
• Prepare the concentrated smears as for a routine blood smear.

For cerebrospinal fluid (CSF)

• Always collect CSF into a tube without anticoagulant.
• CSF requires special handling because of low cell and protein concentrations.
• Please contact the laboratory for hints about handling CSF samples.

Remember:

• Smears should be allowed to air dry; fixation is not required and should be avoided.
• Label tube with the animal’s name, owner’s name, and collection date.
• Label slides with the animal’s name, owner’s name, specimen source, and indication of whether the smear is from direct or concentrated fluid.
• Slides should be kept at room temperature and protected from dust, scratches, or breakage.
• Rigid plastic slide holders work best. Avoid using cardboard slide holders unless shipped inside a rigid box rather than an envelope.
• Fluid should be kept refrigerated until shipping. (Do Not Freeze)
• Ship fluid with an ice pack. Avoid direct contact with ice pack.
• If smears are shipped with the fluid, make sure to seal the smears and protect them from the ice pack.
• For synovial fluid submissions, fluid from each joint should be submitted as a separate cytology, each with a separate submittal form, if complete fluid analysis is requested.
  

Most samples are collected by fine needle biopsy using a fine gauge needle (21-guage or smaller). Larger needles may be helpful for poorly-exfoliating tissue, but may cause more hemorrhage. The mass should be stabilized and the needle inserted into the tissue—with or without an attached syringe. The needle is redirected into the tissue several times (if the mass is large enough) using rapid forward motions to cut cores of tissue. If a syringe is used, gentle negative pressure may be applied to help hold tissue in the needle. Use of suction should not replace forward cutting motions, and excess suction often yields samples that are more difficult to evaluate because of increased blood contamination. When used, negative pressure is released prior to removing the needle from the tissue. Material does not have to be visible in the syringe to have an adequate sample.

Immediately after removal from the tissue, the material in the needle should be expressed onto clean microscope slides by placing the tip of the needle against the slide (not spraying droplets from above the slide).

• Place material close to the frosted end of the slide.
• Place a second slide flat on top of the first slide to gently contact the sample (using gravity only) and immediately spread it toward the center of the slide.
• All samples must be quickly spread before they clot or dry.
• Label slides with the animal’s name, owner’s name, and specimen source.
• Slides should be kept at room temperature and protected from dust, scratches, or breakage.
• Rigid plastic slide holders work best. Avoid using cardboard slide holders unless samples are to be mailed in a rigid box rather than an envelope.
• Do not allow smears to come in contact with formalin fumes. If sent in the same box as a formalin-fixed sample, both samples should be separated into their own zip top bags.

NOTE: Gentle spreading is critical to provide an optimal zone that is thin enough to allow evaluation of intact cells. If material is not spread well, the smears are often too thick to allow adequate morphologic evaluation of the cells. If the drop of material is large, such that a smear will be thick or extend off the slide, part of the drop should be picked up on the edge of another slide and spread onto a third slide with a gentle squash technique. This can be repeated to make several good smears rather than one thick smear.

NOTE: Order one cytologic evaluation per organ or tissue except for lymph nodes.

• You can include samples from multiple lymph nodes on one submission form, but clearly label slides with collection site.
• For all other samples, each organ or tissue requires a separate submission form and must be ordered separately.
• Multiple collections from a single organ can be included on a single form. Label slides as to site.

All fluid cytology requests with sufficient volume include:

• Additional slide preparation from submitted fluid
• Microscopic evaluation of all direct and/or concentrated slide preparations by a clinical pathologist
• Cytology report
• Sample
• Microscopic Description
• Interpretation

In addition to the above:

• Fluid from enclosed spaces (e.g., pleural cavity, peritoneal cavity) will include:
  • Nucleated cell concentration
  • Total protein (TP) by refractometer
  • Spun Hct (PCV) if result is greater than 2%
• Synovial Fluids will include:
  • Nucleated cell concentration
  • Spun Hct (PCV) if result is greater than 2%
  • Total protein (TP) concentration by refractometer
  • Viscosity
  • Turbidity
• CSF will include:
  • Nucleated cell concentration
  • RBC concentration
  • Protein concentration measured biochemically

NOTE: Whenever possible, cell concentrations are measured using an automated hematology analyzer. If that is not possible, cells are counted microscopically using a hemacytometer. If a sample contains a clot, cell concentrations will be measured whenever possible; however, the resulting nucleated cell concentration will be falsely decreased and should be considered a minimum value.