Animal Health Diagnostic Center

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Clinical Pathology  

Clinical Pathology


Clinical Pathology

Sample Submission for Flow Cytometry

Tips on sample submission for immunophenotyping

*If your sample is formalin-fixed tissue (biopsy sample), please contact Michelle Armstrong (mla21@cornell.edu).

Flow cytometry is typically used for the following:

  • Differentiation between B and T cell lymphoid neoplasms
  • Prognostication in lymphoma
  • Differentiation between acute myeloid and lymphoid leukemia
  • Differentiation between reactive and neoplastic expansions of lymphocytes
  • Identification of specific lymphocyte subsets

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Venous blood for flow cytometry (tests: flow leukemia, flow lymphoma, customized flow panels)

  • Dogs and cats (limited panel)
  • 1 ml blood (minimum) in EDTA
  • Fresh is best!! Submit ASAP and keep cool.
  • Important! Submit 2 freshly made unstained blood smears with EDTA.
  • Ideally, request a hemogram or please provide results from a recent hemogram (within last three days).

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Bone marrow for flow cytometry (tests: flow leukemia, flow lymphoma)

  • Usually done concurrently with blood (often blood is diagnostic alone and is preferred if the patient is leukemic)
  • Dogs and cats (limited panel)
  • 1 ml blood (minimum) in CPD (citrate phosphate dextrose; obtained from an unused blood transfusion bag) or ACD (acid phosphate dextrose; yellow top tube)
  • Fresh is best!! Submit ASAP and keep cool.
  • Important! Submit 3 or more freshly made rapidly dried unstained bone marrow smears with the sample for routine bone marrow examination.
  • Ideally, submit blood for a hemogram (with 2 freshly prepared blood smears) or please provide results from a recent hemogram (within last three days).

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Body cavity fluids for flow cytometry (test: flow lymphoma)

  • Usually used for identifying neoplastic lymphocytes (lymphoma) in effusions
  • Dogs and cats (limited panel)
  • 1 ml of fluid in EDTA: The more the better (if nucleated cell counts in the fluid are low, we can only apply 1-2 antibodies)
  • Fresh is best!! Cells rapidly deteriorate in fluid samples, particularly if low protein (can add a few drops of species specific serum if protein is <3.0 g/dL on the refractometer)
  • Important! Must be accompanied by routine cytologic assessment of fluid (so we ensure the cells of interest are in sufficient numbers and adequately preserved for analysis). At the minimum, provide 2-3 rapidly dried unstained smears of the fluid (specify if concentrated or unconcentrated) for morphologic evaluation using Wright’s stain and a copy of the cytology report from the sample. Ideally, request a cytology smear or body cavity fluid analysis from the sample.

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Aspirates for flow cytometry, e.g. lymph node, liver, spleen, mediastinal mass (test: flow lymphoma)

  • Dogs and cats (limited panel)
  • Usually used for identifying neoplastic lymphocytes (lymphoma) in solid aspirates of tissue.
  • Prepare the tube: Add 1 ml physiologic saline (0.9% NaCl, lactated Ringers solution) to a red top tube. Add 0.1 ml of serum from the patient or a different patient of the same species to the tube.
  • Using a 20 g needle and 5 ml syringe, aspirate the affected organ, redirecting the needle (without withdrawing from the tissue) several times if possible.
  • Expel the contents of the syringe and needle into the fluid, gently aspirating the fluid into the syringe several times to retrieve all cells. The ideal sample should be cloudy (indicating a good cell yield), but not too bloody.
  • Fresh is best!! Submit ASAP (keep cool).
  • Important! Must be accompanied by routine cytologic assessment of the sample. At the minimum, provide 2-3 direct unstained rapidly dried smears from the aspirated organ (from the tube or a different aspirate) for morphologic evaluation using Wright’s stain and a copy of the cytology report from the sample. Ideally, request a cytology smear from the sample.

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General pointers on sample submission

  • Expected turnaround time: 48 hours from the day of analysis. We are routinely performing flow cytometry on Tuesday and Thursday.
  • Results: We usually provide results in the form of a cytology report. We also provide the results of the flow cytometric analysis in pdf format.
  • Animals should preferentially have not been treated with any chemotherapeutic agents, including corticosteroids, prior to using flow cytometry. Antigens can be downregulated on lymphocytes after treatment.
  • We never interpret immunophenotyping results in isolation. This is why we request smears for routine cytologic assessment using a Wright’s stain for all immunophenotyping requests.
    • We evaluate the smears to ensure that immunophenotyping is worthwhile on the submitted sample.
    • We need these smears for evaluating cell morphology and other features, which can give us clues as to the diagnosis.
    • We use these smears, along with other clinical pathologic and historical data (signalment, clinical signs, imaging findings), when interpreting immunophenotyping results, so please give us as much information as possible.
  • Cells and antigens deteriorate with age, therefore the most reliable results are obtained on fresh samples.
  • Samples will not be diagnostic if there are insufficient numbers of the neoplastic cells in question or the cells have deteriorated or lysed. This particularly applies to aged fluid samples.
    • Samples of cerebrospinal fluid are usually of too low cellularity to perform immunophenotyping using flow cytometry.
    • In our experience, flow cytometry does not work well on samples with suspected histiocytic sarcoma (the cells lyse readily during the procedure).
    • Before application of the antibodies for flow cytometry, we usually check the sample for sufficient preservation and number of cells in question. If there are insufficient cells or the cells are mostly lysed, we will not proceed with the full flow cytometric evaluation. However, substantial effort and time has been expended to get the sample to this point and, thus, a minimum charge of $40 will be applied to the sample, regardless of whether the complete analysis is done.

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