Flow Cytometry Specimen Requirements

Bone Marrow: 2-7mL heparinized bone marrow. Specimen may be inoculated into a sodium heparin tube (dark green), ACD tube (type A or B), or EDTA tube. Deliver to lab immediately (within 12 hours) and transport at room temperature (20-25 °C). Do not refrigerate.

For overnight storage the specimen may be inoculated into a 50mL conical tube containing RPMI media. Refrigerate (2-8 °C) and transport on “wet” ice. Do not send on dry ice and DO NOT FREEZE.

Peripheral Blood: 10-20mL collected in sodium heparin tube (dark green), ACD tube (type A or B), or EDTA tube. Deliver within 24 hours of collection. Transport at room temperature (20-25 °C). Do not refrigerate.

Solid Tissue/FNA: 5mm solid tissue specimen cut aseptically into small pieces and placed in RPMI media and stored at 2-8 °C. FNA specimens may be placed in RPMI media and stored at 2-8 °C. Transport on “wet” ice. Do not send on dry ice and DO NOT FREEZE. The specimen must not be fixed. If the RPMI media has changed color from a salmon pink to yellow orbright pink – DO NOT USE. If the color has changed it indicates the pH (yellow = acidic, bright pink = alkaline) has changed and the media must not be used. Do not use if the RPMI is cloudy. This would indicate bacterial contamination.

Body Fluids: Volume required for testing depends on the cellularity of the specimen.

The specimen can be transported at room temperature (20-25 °C) and must be stored at 2-8 °C.

Process and Holds: Occasionally a physician may submit a specimen for possible immunophenotyping, depending on the results of permanent sections expected at a later time. In these cases, please write “Process and Hold” on the requisition. The specimen will be processed and held for 2-3 days, during which time the physician must contact thelaboratory to request immunophenotyping.

Specimen Rejection: A specimen may be rejected if it is frozen or fixed, too old, or if it is hemolyzed or clotted. All specimens MUST be labeled with at least the patient’s name and date of collection. Other identifying information is useful, such as hospital number.

Specimen Requisition: The specimen requisition must be completed with the following information:

Patient Name
Date and time of collection
Specimen source or type (ie. Bone marrow, left cervical lymph node, etc…) - Please be as specific as possible.
Ordering physician and contact phone number
Hospital Number or Accession Number (if available)
Patient’s date of birth
Social Security Number (if available)
Thorough clinical history

All insurance information must be included to ensure proper billing.

Panels performed:

There are seven basic panels that are run:

Tissue Panel: Focuses on T and B cell lymphoma, but able to identify non-hematolymphoid neoplasms, granulocytic sarcoma and plasmacytoma.

Bone Marrow Core Panel: Assesses all major bone marrow cells lines and gives the ability to identify abnormality in any line.

CLL/Lymphoma in Bone Marrow Panel: Focuses on T and B cell lymphoma for staging bone marrows, but able to identify aberrant plasma cell populations, myeloid disorders and aberrancies in monocytic cells.

Multiple Myeloma Panel: Focuses on plasma cell evaluation, but able to identify myeloid and lymphoid aberrancy.

Acute/Chronic Myeloid Leukemia Panel (AML/CML): This panel includes markers to assess differentiation of leukemic blasts, common patterns of immunophenotypic aberrancy, and maturation of all cell lines. This panel includes intracellular markers for TdT and myeloperoxidase when necessary.

Acute Lymphocytic Leukemia (ALL) Panel (adult and pediatric): This panel includes markers to assess differentiation of leukemic blasts, common patterns of immunophenotypic aberrancy, and maturation of all cell lines. This panel includes intracellular markers for TdT and myeloperoxidase when necessary.

Paroxysmal Nocturnal Hemoglobinuria (PNH) (peripheral blood only): Focuses on assessment of GPI linked surface proteins.

Methodology:

Each specimen is manually processed. A smear (peripheral blood or bone marrow) or touch prep (tissue) slide is made from the original specimen. A cytospin slide is made from the final cell suspension. The slides are stained and evaluated by the pathologist. A panel of markers is then chosen based on cell morphology and clinical history. Antibody panels are stained and acquired on the flow cytometer. The pathologist analyzes each case using state of the art 4-color (six parameter) flow cytometric immunophenotyping. This method allows for the detection of neoplastic cells, which often express abnormal combinations of antigens.

Hematopathology Requisition>>