Leukemia Immunophenotyping

CPT Code(s)

88184, 88185 x 30, 88189

Synonym(s)

Leukemia Panel

Performed

In-House

Methodology

Flow Cytometry Immunophenotyping

Panel/Profile Components

CD2, CD3, CD4, CD5, CD7, CD8, CD10, CD11b, CD11c, CD13, CD14, CD15, CD16, CD19, CD20, CD22, CD23, CD25, CD33, CD34, CD41a, CD45, CD56, CD57, CD71, CD117, CD138, FMC-7, HLA-Dr, Kappa, Lambda.

Critical Values

N/A

Specimen Collection / Handling Requirements

See Minimum Specimen Requirements.

Minimum Specimen Requirements

For Blood and Bone Marrow Aspirates:

  1. Collection
    1. Collect bone marrow or blood aseptically into a sterile K3EDTA (lavender top) blood collection tube. (ACD or heparin is acceptable anticoagulants if K3EDTA is not available.)
    2. The primary specimen container must be labeled with at least two patient identifiers.
      1. If fewer than two identifiers are visible then request the sending facility to recollect the specimen.
      2. If recollection is not possible, document how the labeling error is addressed in the electronic QA log.
  2. Requirements
    1. A minimum of 1ml of bone marrow or blood is required.
    2. The sample must come with a request specifying the test required, the requesting doctor and facility, and appropriate patient demographic information (name, date of birth or age, identification number, specimen source, and provisional diagnosis). If the test was ordered verbally, additional tests are needed, or if the requisition does not accompany the specimen, the Flow lab will contact the facility immediately and request that the documentation be electronically sent or faxed as soon as possible.
      1. The laboratory personnel receiving the verbal or phone orders must read back the entire order to verify accuracy of transcription. Document who placed the order and the date the order was placed followed by the initials and date of the lab personnel performing the read back.
      2. If the accompanying requisition is unclear, for whatever reason, contact the requesting facility upon receipt of the specimen for further clarification
  3. Handling
    1. The anticoagulated bone marrow must be stored at room temperature (200 to 250C), no longer than 24 hours prior to staining.
  4. Transportation
    1. Maintain and transport bone marrow samples at room temperature (200 to 250 C)
    2. Transport samples in a reseal-able plastic biohazard bag.
    3. Place the biohazard bag(s) containing the samples in a padded carrying case or a Styrofoam box. Do not place biohazard bags in a garment pocket for transport.
    4. Avoid drastic temperature changes, such as <100C or >370C.
      1. In hot weather, pack specimens in a container which has insulating material around it and place this container inside another that contains a cold pack (ice pack) and some type of absorbent material. This will help maintain the specimen at ambient temperature. NOTE: DO NOT PLACE BONE MARROW SPECIMENS (OR BLOOD) DIRECTLY ON ICE OR REFRIGERATE. DO NOT FREEZE.

For Tissue

  1. Collection
    1. Place the lymphoid tissue in a conical tube with RPMI medium. Tissue for flow cytometry should only be collected after tissue has been submitted for histology in formalin or BS fixative.  The tissue sample that will be submitted to the flow lab must not be fixed in formalin or similar fixative.
    2. The  primary  specimen  container  must  be  labeled  with  at  least  two  patient identifiers.
      1. If fewer than two identifiers are visible then request the sending facility to recollect the specimen.
      2. If recollection is not possible, document how the labeling error is addressed in the electronic QA log.
  2. Requirements
    1. A minimum of 0.5 cm3 of tissue is required. If less tissue is available, consider freezing it for frozen section immunostains or molecular studies. Refer any questions to the Supervisor.
    2. The sample must be accompanied by a request specifying the test needed, the requesting doctor and facility, and appropriate patient demographic information (name, date of birth or age, identification number, specimen source, and provisional diagnosis). If the test was ordered verbally or if additional tests are needed, or if the requisition does not accompany the specimen, the lab will contact the facility and request that documentation be sent or faxed as soon as possible.
      1. The laboratory personnel receiving the verbal or phone orders must read back the entire order to verify accuracy of transcription. Document who placed the order and the date the order was placed followed by the initials and date of the lab personnel performing the read back.
      2. If the accompanying requisition is unclear, for whatever reason, contact the requesting facility upon receipt of the specimen for further clarification.
  3. Handling
    1. The lymphoid tissue is usually at room temperature and brought immediately to the lab for processing.
    2. If there is a delay in sending the lymph tissue, it must be stored at 4O C in RPMI, no longer than 24 hours before staining.
  4. Transportation
    1. Maintain and transport lymphoid tissue samples at room temperature (20O to 25OC) stored in a conical tube containing RPMI.
    2. Transport samples in a sealed plastic biohazard bag.
    3. Place the biohazard bags containing the samples in a padded carrying case or a Styrofoam box. Do not place biohazard bags in a garment pocket for transport.
    4. If samples cannot be immediately delivered to the lab for processing, place tissue in RPMI and place on wet ice or in the refrigerator.
    5. NOTE: DO NOT FREEZE OR FIX LYMPH TISSUE.
    6. Samples from University Hospital:
      1. Samples are picked up from Histology along with a request for lymphocyte studies.
      2. Ensure the surgery number is correct when signing sample out on Histology Log before leaving.
      3. Initial for the sample being picked up.
      4. Place samples in small carrying case and transport to flow lab.

For All Body Fluids and Fine Needle Aspirates (FNA):

  1. Collection
    1. Collect spinal fluid (CSF) in a sterile container
    2. Collect pleural fluid/ascitic fluid in a ratio of 1 ml of sodium heparin or ACD to 100 ml of fluid.
    3. Collect FNA specimens in a sterile container that contains RPMI
    4. The  primary  specimen  container  must  be  labeled  with  at  least  two  patient identifiers.
      1. If fewer than two identifiers are visible then request the sending facility to recollect the specimen.
      2. If recollection is not possible, document how the labeling error is addressed in the electronic QA log.
  2. Requirements
    1. CSF, Body fluid, and FNA samples must have an adequate amount of cellularity present in order for proper flow cytometric analysis. Minimum acceptable volumes are dependent on the overall cellularity of the available specimen.
    2. The sample must be accompanied by a request specifying the test needed, the requesting doctor and facility, and appropriate patient demographic information (name, date of birth or age, identification number, specimen source, and provisional diagnosis). If the test was ordered verbally or if additional tests are needed, or if the requisition does not accompany the specimen, the lab will contact the requesting facility and request that documentation be sent or faxed as soon as possible.
      1. The laboratory personnel receiving the verbal or phone orders must read back the entire order to verify accuracy of transcription. Document who placed the order and the date the order was placed followed by the initials and date of the lab personnel performing the read back.
      2. If the accompanying requisition is unclear, for whatever reason, contact the requesting facility upon receipt of the specimen for further clarification.
  3. Handling
    1. The freshly collected body fluid or FNA is brought immediately to the lab for processing.
    2. If there is a delay in sending the specimen, it must be stored at 4O C, for no longer than 24 hours before staining.
  4. Transportation
    1. Maintain and transport the body fluid at room temperature (20O to 25O C) if delivery will take less than 1 hour. Transport on ice if further delay is expected.
    2. Transport samples in a re-sealable plastic biohazard bag.
    3. Place the biohazard bag(s) containing the sample(s) in a padded carrying case or a Styrofoam box. Do not place biohazard bags in a garment pocket for transport.
    4. If samples cannot be immediately delivered to the lab for processing, store the tube on wet ice or in the refrigerator. NOTE: DO NOT FREEZE OR FIX FLUID.

Turnaround time

Results to Client: 5 business days

Communication

  • Turnaround time non-conformity: Email or call Pathologist on request form to notify them of the delay
  • Specimen rejection: Cause for specimen rejection is noted after the sample has arrived at UT Health San Antonio and the Pathologist on the request form or applicable personnel will be contacted by email or phone.
  • Technical Updates: Email Applicable Laboratory Director.

Quality

For all specimens

  1. The diagnosis of malignant lymphoma or leukemia is made on the identification of an abnormal population of cells, i.e., an aberrant phenotype or monoclonality. This is done by comparison of the staining of different markers in the same specimen. Therefore normal values are not used in the interpretation.
  2. The patient’s name and number are verified on the receiving system by two persons.
  3. Notify signing pathologist of specimens that have a viability of 50% or less.
  4. All new monoclonal antibody lot numbers are verified with previous lot numbers.
  5. Reactivity of the Lyse is assessed daily by visually inspecting each tube for clear supernatant without sediment of intact RBC after centrifugation.
  6. If supernatant remains red, a fresh new batch of lysing solution may be required to re-lyse the patient sample.
  7. Reagent-grade water and de-ionized water free of extraneous contamination.
  8. T & B cell comparisons:
    1. Normal lymphocytes are marked with T cell, B cell or LG/NK cell markers.
    2. The total T cell population should compare with the total for all subsets of T (i.e., T cytotoxic/suppressor plus T helper/inducer should be equal to the total T).
    3. Compare all Pan T-cell markers (CD3, CD2, CD5, and CD7) and Pan B-cell markers (CD19, CD20, and KAPPA/LAMBDA).
  9. CAP Survey: Performed the same way as patient samples.