CLINICAL FLOW CYTOMETRIC ANALYSIS
Main Laboratory, 1st floor

Director: Ila Bansal, M.D. (501-686-6230)
Supervisor:  Linda Whigham, AIBMS (501-686-5953)
Technical Specialist: Rebecca Owens, BSMT (501-686-5953)
Hours: Monday through Friday, 8:00am - 5:00pm

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Flow Cytometric Analysis of Cell Surface Antigens (Immunophenotype)

Clinical Significance:
Changes seen in lymphocyte subsets, such as T or B cell populations, can be indicative of immunological changes produced by various diseases or treatments.  The Helper/Suppressor Panel is used as a general screen for all lymphocyte subsets in cases where there is no knowledge of the underlying cause or disease which may result in the patient's health problems.  Acquired Immune Deficiency Syndrome (AIDS) has several clinical symptoms, one of which is an abnormal decrease in the T helper cell population or CD4 in the peripheral blood.  This abnormality can show up early in the disease and can be followed during the course of the disease and during various treatment programs as a monitor of the progression of the disease or the effectiveness of the treatment.  After a bone marrow transplant, lymphocyte subsets are monitored using the Immune Studies panel to determine the status of certain relevant subsets.

Organ and bone marrow transplants have fast become a well established treatment for patients with chronic life threatening diseases.  Flow cytometry has proven to be a valuable aid in evaluating these patients for disease remission, transplant engraftment or rejection and for response to growth factors (CD34) or immunotherapy (CD3).

The diagnoses of leukemia and lymphoma have, until recently, consisted of evaluating the specimen by various morphological and histochemical criteria.  More extensive immunophenotyping panels by flow cytometry are used to distinguish Chronic Lymphocytic Leukemia from a generalized increase of lymphocytes (Chronic Leukemia/Lymphoma Panel) or to distinguish Acute Lymphoid Leukemia from Acute Myeloid Leukemia (Acute Leukemia Panel).

Flow cytometry is not indicated for use in the diagnosis of Chronic Myeloid Leukemia in the chronic stage.  Flow cytometry can also only be used to rule out Non-Hodgkin's Lymphoma but not to diagnose Hodgkin's Lymphoma.

Cylex is a non-flow cytometric assay performed in the Flow Cytometry lab.  Cylex, an immune function assay that detects cell-mediated immunity status of CD4 cells, can be used as an additional test for monitoring immunosuppressed patients.  This test can be used in conjunction with CD4 enumeration or in place of the CD4 count.  Discussion of this test and specimen requirements for it are discussed at the end of the discussion of Flow Cytometry assays.

I.    Lymphocyte Subset Analysis for evaluating immunological status.

• Helper/Suppressor Cell Panel (H/S)
  Consists of evaluation (% positive and absolute numbers) of total T cell (CD3), Helper cell (CD4), Suppressor cell (CD8), total B cells (CD19), CD4/CD8 ratio, Natural Killer Cells (CD16, CD56), as well as WBC and absolute lymphocytes.

• CD4 T Cell Subset (CD4)
  Consists of evaluation of CD4 Helper cell subset.  This test is used mainly for evaluating and monitoring HIV positive patients.  A percent positive and absolute CD4 count, WBC and absolute lymphocyte count are reported.  No other subsets are reported.

• Immune Studies Panel (IMSPB)
  Consists of evaluation of two T cell markers (CD3 and CD4) and one B cell marker (CD19).  This panel is used only for monitoring patients undergoing bone marrow transplants with peripheral stem cell products and chemotherapy.

II.    Chronic Leukemia/Lymphoma and Acute Leukemia Phenotype

        The following tests do not have SoftLab test codes.  These Flow Cytometry tests should be ordered by panel name either in
        E-Chart or on the requisition sheet.  Results can be found in SOFTPath.

• Chronic Leukemia/Lymphoma Panel
  Enumeration of all lymphocytes and subsets as an aid in distinguishing leukemia/lymphoma from benign lymphocytosis. Panel consists of various differentiation markers found on T and B cells that can be used to detect monoclonicity or abnormalities associated with leukemia/lymphoma.  Should be used in conjunction with other diagnostic tests.

• Acute Leukemia Panel
  Enumeration and identification of blasts with T cell, B cell, and myelo/monocytic cell markers.  To be used in conjunction with morphological diagnosis and/or special histochemical stains to distinguish Acute Myeloid Leukemia (AML) from Acute Lymphoid Leukemia (ALL).

• Cytoplasmic Immunoglobulins (CIg) vs DNA
  This assay is used to distinguish normal plasma cells from malignant plasma cells by determining the monoclonicity of the cells in conjunction with the DNA content of the cells.

• Plasma Cell Phenotype
  A panel of antibodies is used to identify and classify malignant plasma cells.  This assay is not routine and is ordered as needed.

• Pathologist Review
  A request for a pathologist review of a prepared differential of the specimen with a follow-up order by him/her for the appropriate panel may be ordered in place of any of the above tests.

III.    Transplantation Panels:

• CD3 Lymphocyte Levels (CD3/5)
  Immunosuppressive therapy, used in solid organ transplants, causes a disappearance of  T cells. Failure to respond to therapy is demonstrated by a slow but steady rise in the T cells.  CD3 and CD5 antigens are used as a monitor of this therapy.

• CD34 (Human Progenitor Cell Antigen) Levels (APB34){ProCount}
  The CD34 antigen is expressed on stem cells that are necessary for reconstituting the bone marrow. Bone marrow transplant patients undergo a series of procedures that enhance CD34 production, collect CD34-enriched products, destroy the diseased bone marrow and then return the CD34 enriched products back to the patient. Flow cytometry monitors the efficiency of these procedures by determining CD34 levels in blood and stem cell products.

General Specimen Information

Specimen Requirements for Flow Cytometry Tests: 

          Peripheral Blood for  CD4, Immune Studies, Helper/Suppressor Panels
          1 EDTA (Lavendar Top)

Peripheral Blood (for all other tests)
1 sodium heparin tube (Green Top)
1 EDTA tube (Lavender Top)

Bone Marrow Aspirate
Orders for flow cytometry tests should be made when arrangements are made for the bone marrow procedure.

Biopsies and Solid Tissues
Orders for flow cytometry tests should be made when arrangements are made for the biopsy or surgical procedure through the Department of Pathology or Radiology.

Body Fluids
All body fluids should be obtained and held at room temperature before being transported to the laboratory.  The specimens should be received in the laboratory within 24 hours of being obtained from the patient.
 

Specimen Rejection:

1. Clotted tubes
2. Collected in improper tube
3. Partial draw
4. Insufficient number of viable cells
5. Specimen older than 72 hours
    

Hours of Operation:
Samples are accepted through the Clinical Laboratory Specimen Receiving Area at all hours.  All specimens must be received within 24 hours of collection.  The regular work hours for Flow Cytometry are 7:30am to 5:30pm Monday through Friday.  Any specimen received outside of these hours will be held until the next regular work day.  Contact the Flow Cytometry lab during normal working hours at 501-686-5953 for additional instructions for after-hour specimens.


Results Turnaround Time

• H/S, CD4, IMSPB, CD3, APB34{ProCount}:  4 hours to 24 hours, during regular hours of operation. (See Hours of Operation, above)
   
• Chronic Leukemia/Lymphoma Panel, Acute Leukemia Panel, CIg vs DNA and Plasma Cell Phenotype requires a written interpretative report (SoftPath) from a pathologist along with the qualitative results.  An initial verbal report may be given to the requesting physician before final results are available.  Final interpretive report turnaround time is 24 to 48 hours.
  
  
   

Non-Flow Cytometric Assay

CYLEX  Assay

ImmunKnow(tm) - the Cylex Immune Cell Function Assay detects cell-mediated immunity (CMI) in an immunosuppressed population by measuring the concentration of ATP from CD4 cells following stimulation.  The amount of ATP present in stimulated cells is a measure of lymphocyte activity and function.  Response to immunosuppressive therapy varies among individuals; therefore, an assessment of a patient's immune cell function may provide useful information to the clinician in the course of individual patient management.  This measurement is made on heparin anti-coagulated whole blood using a luminometer and luciferin/luciferase.

General Specimen Information:
Request Procedure:  Samples are accepted through the Clinical Laboratory Specimen Receiving Area.  Due to the nature of the assay, please make note of the limited availability of the assay.  Specimens are accepted Monday-Wednesday, and Thursday (before
Noon).  The test is not available on Fridays, weekends, holidays or the day before a holiday.  All tests ordered outside of the accepted days will be cancelled and a redraw requested.

Specimen Requirements for Cylex Test:

Peripheral Blood:  1 sodium heparin tube (Green Top), specimen must be less than 30 hours old at the time of assay.

Results Turn-a-Round Time:  24 hours

Table of Tests Performed in Flow Cytometry

Test Name (TEST CODE)	Specimen Requirements	Therapeutic Range	Expected Turnaround Time
CD3 Lymphocyte Levels (CD3/5)	1 Green Sodium Heparin top + 1 Lav. top	Normal ranges: CD3:  68-80%; 1117-2250/µl CD5:  70-82%; 700-3280/µl Therapeutic range CD3 or CD5 = < 10%;  < 100/µl	4-24 hrs
CD4 T Cell Subset (CD4)	1 Lav top	39-57%, 720-1348/µl	4-24 hrs
CD34 [Human Progenitor Cell Antigen (APB34) {ProCount}	1 Green Sodium Heparin top + 1 Lav top	Normal range = Negative Therapeutic range is any increase over the normal range.	4-24 hrs
Helper/Suppressor Cell Panel (H/S)	1 Lav top	CD3:  64-82%, 1171-2005/µl CD4:  39-57%, 720-1348/µl CD8:  17-31%, 318-710/µl CD19:  8-16%, 151-343/µl CD16/56:  7-21, 145-453/µl H/S Ratio 1.0-3.6	4-24 hrs
Immune Studies Panel (IMSPB)	1 Lav top	CD3:  64-82%, 1171-2005/µl CD4:  39-57%, 720-1348/µl CD19:  8-16%, 151-343/µl	4-24 hrs
Chronic Leukemia/Lymphoma Panel	1 Green Sodium Heparin top + 1 Lav top, Bone marrow aspirate, Biopsy, Tissue or Body Fluid	Interpretive report.  Includes enumeration of all lymphocytes and subsets.	24-48 hrs
Acute Leukemia Panel	1 Green Sodium Heparin top + 1 Lav top, Bone marrow aspirate, Biopsy, Tissue or Body Fluid	Interpretive report.  Includes enumeration of blasts with T cell, B cell and myelo/monocytic cell markers	24-48 hrs
Cytoplasmic Immunoglobulins (CIg) vs DNA	1 Green Sodium Heparin top + 1 Lav top, Bone marrow aspirate, Biopsy, Tissue or Body Fluid	Interpretive report.	24-48 hrs
Plasma Cell Phenotype	1 Green Sodium Heparin top + 1 Lav top, Bone marrow aspirate, Biopsy, Tissue or Body Fluid	Interpretive report.	24-48 hrs.
Cylex Immunknow Assay (CYLX)	1 Green Sodium Heparin top Collect specimens Mon - Wed or Thurs before noon.  Do not collect on Friday, weekends, holidays, or the day before holidays. Specimen must be less than 30 hours old to be tested.	ATP: Low <225 ng/mL Moderate 226-524 ng/mL High >525 ng/mL	48 hours

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