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Flow Cytometry - Leukemia/Lymphoma

About the Testing

Flow based immunophenotypic testing represents an accurate and invaluable tool for the clinician and the pathologist in the diagnosis, subclassification, prognostication and follow up (MRD) of acute and chronic leukemias as well as non-Hodgkin lymphomas.

 Leukemia/Lymphoma Test List

Collection Guidelines for Leukemia/Lymphoma Panels

Please note: Bone Marrow Media (BMM) and Tissue Transport Media (TMM) are only made available for Calgary Zone collections.

1. Tissue/Lymph Node

  • Obtain 2-3 mm thick cross section of fresh, unfixed lymph node. 
  • Place in Tissue Transport Medium (TTM). If TTM is not available, use normal saline.
  • DO NOT send directly to Flow Cytometry. Immediately deliver sample together with the specimens destined for histological processing to Anatomical Pathology. For samples collected outside of Alberta, please place specimens in RPMI for transport to Calgary Laboratory Services.  
  • Any samples submitted for Flow Cytometry analysis MUST NOT be put into cytology fixative.
  • Do not freeze specimens.
  • Ultimately, the decision on how much and if any tissue or body fluid should be sent for immunophenotypic analysis lies in the hands of the referring pathologist/cytologist supervising the processing of the specimen at the referring hospital site.

2. Peripheral Blood

For samples collected within Alberta:
Flow Cytometry testing ordered by non-Hematologists will be screened from a current (or within the last 3 months) CBC/DIFF/PBS result, and tested only when one or more of the following criteria are met:

  1. Lymphocytoses > 3.0 x 109/L
  2. Monocytoses > 1.0 x 109/L
  3. Thrombocytopenia < 100 x 109/L
  4. Blasts present
  5. Flow Cytometry testing recommended from a previous PBS report
  • Collect in 8.5 mL yellow top ACD "A" tube. Partial draws are not recommended as the anticoagulant is hypotonic,  Peripheral blood samples in ACD "A" should optimally be tested within 24 hours. Some samples may be stable up to 5 days when stored at 4°C.
  • ACD "B", EDTA, and heparin are also acceptable if ACD "A" is not available.  EDTA samples must be tested within 24 hours and heparin samples must be tested within 48 hours when stored at 4°C.
  • Hemolyzed, frozen, or clotted samples are not accepted.
  • Whenever possible, the peripheral blood requiring flow cytometric analysis should be accompanied by a peripheral blood smear. This will provide morphologic correlation for accurate interpretation of the immunophenotypic data.

 3. Bone Marrow

    • 1 mL of bone marrow aspirate should be collected in 3 mL of Bone Marrow Media (BMM) available from Flow Cytometry (403-944-4771).  The media may be kept frozen at –70°C until needed. Ensure the BMM is completely thawed before adding to sample. BMM may be stored at 4°C for 1 week after thawing.
    • Any samples submitted to Flow Cytometry MUST NOT be put into cytology fixative.
    • Send samples to the Flow Cytometry lab as soon as possible, however, they may be kept at 4°C overnight if testing is not started on the same day.
    • Hemolyzed or frozen samples are not accepted. Clotted samples may be submitted for testing, however, it is a suboptimal specimen and results may be unreliable. In the event of a dry tap, a core biopsy may be submitted for testing.
    • Whenever possible, a bone marrow smear(s) collected at the bedside, should accompany the aspirate for testing in the Flow Cytometry lab. This will provide morphologic correlation to aid in accurate interpretation of the immunophenotypic data.

    4. CSF

    • Use Flow Cytometry requisition to avoid delays in specimen transport. Include clinical diagnosis and indicate if performing immunophenotyping analysis (lymphoma, acute leukemia). Quantity of specimen required is dependent on cellularity. DO NOT refrigerate or add CytoLyte® solution to these specimens.       
    • CSF: minimum of 2 mL of CSF is required.  10 mL of CSF is preferred to ensure adequate cells for testing. See CSF Collection Guidelines for more information, including Specimen Handling and Transport.            

    5. Body Fluids

    • Includes abdomen, pleural, peritoneal, ascites, thoracentesis, pericardial fluids, and pelvic, cul de sac and gutter washes. Does not include FNAs (Fine Needle Aspirates).
    • Use Flow Cytometry requisitions to avoid delays in specimen transport. Include clinical diagnosis and indicate if performing immunophenotyping analysis (lymphoma, acute leukemia). DO NOT refrigerate or add CytoLyte® solution to these specimens.
    • Sample volume: minimum of 10 mL of body fluid is required. Quantity of specimen required is dependent on cellularity.  
    • Send samples immediately to DSC Cytopathology. DO NOT send directly to Flow Cytometry.
    • Specimens submitted to CLS from outside Alberta: DO NOT centrifuge or freeze samples. Ship at 4°C, using an overnight courier. Please follow Flow Cytometry Shipping and Handling instructions when sending to CLS.

    6. BAL

    • Use Flow Cytometry requisitions to avoid delays in specimen transport. Indicate clinical diagnosis on the requisition.
    • Sample volume: minimum of 1 mL BAL required. 5 mL of BAL is preferred to ensure adequate cells for testing.
      Quantity of specimen required is dependent on cellularity. DO NOT refrigerate or add CytoLyte® solution to these specimens.
    • Send sample immediately to DSC Microbiology. DO NOT send directly to Flow Cytometry.
    • Specimens submitted to CLS from outside Alberta: DO NOT centrifuge or freeze samples. Ship at 4°C, using an overnight courier. Please follow Flow Cytometry Shipping and Handling instructions when sending to CLS.

    7. Fine Needle Aspirates (FNA) /Core Biopsies

    • Unfixed fine needle aspirate/core biopsies should be submitted to Anatomical Pathology together with the specimens destined for histopathologic processing.  A pathologist on duty will be responsible for referring a portion of the obtained tissue for immunophenotypic analysis, where appropriate. DO NOT send directly to Flow Cytometry.        

    Within Alberta Testing Guidelines 

    Flow based testing is not recommended and will be cancelled for the following disease investigations:

    • Neuroblastoma
    • Classic Hodgkin staging bone marrow
    • Diffuse large B cell lymphoma (DLBCL) staging bone marrows (PET scan recommended procedure)
    • Anemia (single cytopenia)
    • Thrombocytopenia (single cytopenia)
    • Rule out Myelodysplastic syndrome (MDS) if CBC does not show cytopenia in more than 1 lineage
    • Myeloproliferative neoplasms (MPN)
      • Chronic eosinophilic leukemia
      • Chronic Myelogenous leukemia (CML)
      • Chronic neutrophil leukemia
      • Essential thrombocythemia (ET)
      • Mastocytosis
      • Polycythemia vera (PCV)
      • Primary Myelofibrosis (MF)