Flow cytometry
Test description
Flow cytometry characterizes cell populations in a sample based on size properties and the proteins expressed on the cell surface. This test helps differentiate a reactive from neoplastic process and provides an immunophenotype of a neoplastic population. For dogs, immunophenotyping by flow cytometry is almost always the test of choice because it can provide both a diagnosis and prognostic information. Cats more often have non-neoplastic lymphocyte expansions in the blood and other organs, and fewer antibodies are available for immunophenotyping.
Flow cytometry is performed on live cells in fluid suspension. The sample must be acquired within 24 hour of shipping and kept cold (but not frozen) to maintain viability.
Species
Canine, Feline
Submission instructions
Blood and bone marrow
- Place at least 1mL of sample in EDTA tube.
- Blood samples must have a current (within two days) complete blood count (CBC). You can either include a copy of your results or check the “do a CBC at CSU” box on the submission form.
- To submit CBC to CSU, please include a second EDTA and fresh blood smear(s). If a second EDTA tube is impossible to obtain we can split a single tube – it is more important to have a properly filled tube for an accurate CBC.
Lymph node, mediastinal, and other organ aspirates
- Place 1ml of saline (.9%, LRS, Norm R) into a no-additive, white-top or red-top tube (plain tube with no additives or serum separator).
- Add .1 ml of serum from the patient, or another healthy animal of the same species, to the saline in the white-top or red-top tube.
- Aspirate using suction.
- Gently squirt contents into the tube containing the saline and serum mixture. Rinse syringe by drawing up saline and serum mixture and gently squirting it back into the tube. Don’t make bubbles. Repeat aspiration and rinsing multiple times until solution is no longer transparent. A turbid solution usually indicates adequate cellularity for flow cytometry testing.
To purchase sample collection tubes for flow cytometry of organ aspirates, complete the sample collection tube order form.
Cavity fluid
If possible, send one EDTA tube and one red-top tube containing 400ul or more of fluid. Send EDTA if only enough for one tube. Add a few drops of serum if the total protein is less than 4 mg/dl.
Special instructions
Send all samples overnight with a cold pack for delivery. Do not freeze samples!
Cost
Polymerase chain reaction for antigen receptor rearrangement (PARR)
Test description
The PARR assay is a PCR assay that amplifies DNA. Because this test is performed on DNA, which is a stable molecule, special handling and shipping is not necessary. PARR can be performed on any samples, including stained and unstained cytology slides and fluids. The PARR assay is also performed on biopsies that have been embedded in formalin and examined by histology. Sample preparation for this testing is different than sample preparation for all other sample types.
We estimate that we need at least 50,000 lymphoid cells for a diagnostic PARR assay.
Species
Canine, Feline
Submission instructions
Blood and bone marrow
- Submit EDTA tube containing at least 500ul of blood or bone marrow sample. 4-5 very cellular bone marrow slides can also be submitted, but please do not submit slides of peripheral blood.
- If available, please provide a complete blood count with a pathology review or cytology/histology report of the bone marrow.
Lymph node and other organ aspirates
- Submit 4-5 cellular slides (smears or cytospins) or fluid in EDTA. No additional fluid needs to be added (i.e. saline/serum). Glued cover-slipped slides cannot be used.
- If you aspirate onto slides and want to confirm that you have a good sample, the slides can be stained and still used for PARR.
- Please provide a copy of relevant cytology and/or fluid analysis with submissions.
Cavity fluid
- Send fluid in EDTA and/or on 4-5 cellular slides (smears or cytospins).
- If you aspirate onto slides and want to confirm that you have a good sample, the slides can be stained and still used for PARR.
- Please provide a copy of relevant cytology and/or fluid analysis with submissions.
Cerebrospinal fluid (CSF)
- Multiple cytospin preps are the best sample (stained or unstained), but fluid can also be sent in an EDTA tube.
- For the sample to be diagnostic we estimate you need 50,000 lymphoid cells. Therefore, if the CSF has a lymphocyte count of 100/ul, we need at least 500 ul of fluid, or the cells from 500 ul spun onto slides.
- Please provide a copy of cytology and/or fluid analysis with submissions.
Formalin-fixed paraffin-embedded (FFPE) tissue
- Cut five curls (or scrolls) from the histology block at 20uM (microns) thick. Please do your best to keep the curls intact in your tube.
- Alternatively, you can send the histology block to CSU and the curls will be prepped by the CSU Histopathology Laboratory.
- Include either a copy of a histology report for the site being tested or a concurrent histology test request.
Special instructions
Non-diagnostic samples are obtained when there is too little lymphoid DNA, or when there is an inhibitor of the PCR reaction. If a cytology report is submitted with the sample and indicates that the sample was highly cellular, we will repeat the assay, but if that is not successful, we will assume there was insufficient DNA or an inhibitor in the sample (mast cells can sometimes inhibit the PARR assay). We try to contact the submitting clinic if we think the sample lacks the cellularity to achieve a diagnostic result, but ultimately it is the responsibility of the submitting clinic to send an adequate sample.
Samples can be sent using any mail delivery service and should be sent at the speed in which you want to receive results.
Cost
Ki67
Test description
Ki67 is a protein that is expressed in the nucleus of a cell during proliferation but is absent when cells are quiescent. It is a commonly used marker of proliferation in immunohistochemistry. In some cancers, high Ki67 expression predicts a more aggressive disease.
Ki67 can help to distinguish cases of B-cell chronic lymphocytic leukemia with a short survival from those with a more indolent clinical course. Ki67 is detected using a flow cytometry assay.
To perform Ki67, the dog must have been diagnosed with B-cell chronic lymphocytic leukemia by the Clinical Hematopathology Laboratory within the last month.
Ki67 testing should be performed prior to treatment.
Species
Canine
Submission instructions
- Prepare and ship samples on ice using flow cytometry guidelines.
- Take Ki67 samples Monday or Tuesday for overnight shipping and receipt Tuesday or Wednesday.
Cost
Cell block
Test description
Cell block techniques can be used to fix fluid samples (i.e. blood, effusions, or aspirates) into a paraffin block that can be used for immunohistochemical staining. This technique can increase diagnostic accuracy by applying additional antibodies that are not readily available for flow cytometry and allow us to investigate expression of intracellular antigens. Immunohistochemistry typically provides clearer and more consistent results than immunocytochemistry.
Species
Canine, Feline
Submission instructions
We may suggest cell block staining on flow cytometry samples. If you would like cell block staining on samples that have not been previously analyzed by flow cytometry, please contact the laboratory.
Blood or effusions
Recommend 1mL of fluid, minimal amount >500uL. Include a fresh blood smear.
Aspirates
Sample must be highly cellular. Recommend resampling for a highly cellular sample if cell block is warranted post flow cytometry analysis. Poorly cellular fluids will not yield an optimal cell block for processing and interpretation. Include fresh smear with your submission.