Anne LeMaistre, M.D.

Assistant Professor

Department of Pathology and Laboratory Medicine

The University of Texas-Houston Medical School

This CME activity was planned and produced in

accordance with the ACCME Essentials.

This document is worth 1 CME credit hour. Total Study

Time = 1 hour.

Educational Objectives:

Upon completion of this activity, The participant should be able to:

• know the basic methodology behind diagnostic flow cytometry
• know how specimens are collected , processed and analyzed by flow cytometry
• understand how different patterns of immunophenotypes relate to specific leukemias and lymphomas
• understand the clinical uses of DNA cell cycle analysis and image analysis

• Introduction
• Immunophenotyping
• Quality Assurance
• DNA Cell Cycle Analysis
• Bibiliography

Test

In fluorescence, the exciting (incident) light is absorbed by the electron, which increases the energy of the electron and raisesit to a higher orbital. This electron is not stable at this higher energy level, however, and quickly reverts to the lower orbital in less than a second. During this reversion, the electron will give off some of its energy as light, which is thefluorescence. Since the amount of energy given up is not as much as input, the wavelength of the fluorescent light is longer (less energetic) than the incident light. The recent development of chromogens that are excitable by the standard argon laser has allowed bench top flow cytometers to routinely perform these analyses. In addition to allowing a more definitive typing of all leukocyte populations, multicolor analysis can decrease the number of tubes needed to run a panel of antibodies.

The recommended method of collection is to collect blood by venipuncture into evacu ated tubes containing an appropriate anticoagulant. Use pediatric tubes for pediatric specimens and be sure the tube is filled until the vacuum is expended. Mix the blood well with the anticoagulant in the tube to prevent clotting. There are three different anticoagulants used in flow cytometry. If the specimens will be processed within thirty hours of collection then any of the three anticoagulants, potassium EDTA, acid citrate dextrose (ACD), or heparin, may be used. If the specimen will not be processed within thirty hours, then use either ACD or heparin.

Specimen Transport and Integrity

Maintain and transport all specimens for flow cytometry analysis at room temperature (18-22 degrees C). This is necessary to maintain the viability of the cells and their expression of antigens. Once the specimen arrives at the laboratory, inspect the specimen and take the following actions if necessary:

• If the specimen feels too hot or cold, but is not obviously frozen or hemolyzed, make a note on the worksheet. Light scatter abnormalities will be noted if the specimen has been harmed.
• Reject the specimen and request a recollection if the specimen is frozen, hemolyzed, over 48 hours old, or clotted.

A hematologic analysis for the white blood cell count and lymphocyte differential should be performed within six hours of collection. Generally this is done on an automated instrument, but if it "flaggs" the lymphocyte population, a manual differential should be performed.

For optimal results specimens should be processed immediately through fixation and the immunophenotype should be performed within 24 hours. The maximum time period for analysis is dependent on the collection agent used for the specimen and should be established by each individual laboratory. A general rule is that specimens can be processed for immunophenotype up to 30 hours after collection, but should not be analyzed after 48 hours. A direct two-color immunoflourescent whole blood lysis method is used (better known as the "stain, then lyse" procedure). Video 1.

A positive control is used to determine whether specimen processing is optimal. Generally a whole blood specimen is used which is obtained from a donor who ideally matches the patient population analyzed in the laboratory. If the control falls outside of the established normal ranges for the laboratory then the reason must be determined. Examples of reasons for positive control failure might include poor lysis or poor labeling.

Generally the highest volume test in a flow cytometry laboratory is a CD4/CD8 analysis of the peripheral blood performed in patients suffering from human immunodeficiency virus (HIV) infection. In acquired immunodeficiency syndrome the HIV-1 infection causes a decrease in the T-lymphocyte population containing the CD4 receptor. The percentage of CD4 positive (CD4+) T-cells circulating in the peripheral blood has been shown to have a direct correlation to the severity of illness in the patient. The lower the percentage of CD4+ cells then the more likely the patient will develop a serious infec tion. For this reason, prognostic indicators and clinical decision points have been established on certain levels of CD4+ T-cells. Therefore, a CD4/CD8 analysis is performed approximately every 3-6 months to closely follow these patients.

The CDC has recommended the following panel to be performed for peripheral blood lymphocyte analysis of CD4/CD8.

Recommended CD4/CD8 Panel

FITC            PE      REASON FOR USING

CD45            CD14    For gating, lymphocytes are brightly positive 
                        for CD45 and negative for CD14
Isotype         Isotype Control to set cursors, or discriminators,
                        for positivity in the samples to follow
CD3             CD4     To measure CD4+ T cells; only CD3+/CD4+ cells
                        should be reported as CD4+ cells 
CD3             CD8     To measure CD8+ cells; only CD3+/CD8+ cells
                        should be reported as CD8+ cells.  CD3-/CD8+
                        cells are natural killer cells
CD3             CD19    To measure B cells to account for all lymphocytes
                        and for quality assurance
CD3             CD16    To measure natural killer cells CD16+/CD56+/CD3-
                CD56    for quality assurance, to account for all lymphocytes

The isotype control serves to detect nonspecific binding of the mouse monoclonal antibody to cells. An antibody with no human specificity is always used, but the antibody should always be of the same isotype as the panel antibodies. Also it is used for setting markers to separate fluorescent negative and positive populations. Besides helping to determine the T cell populations, CD3 serves as an internal control which will demonstrate any tube to tube variability. The CD3 positive results should have only a 3% variability. If the value is greater then 3%, then the antibody combination should be restained and analyzed.

The use of the CD3/CD19 is to determine the number of B cells.

The measurement of CD4+ cells is reported as a percentage and an absolute number. To calculate the absolute CD4+ T-cells it is necessary to perform a white blood cell (WBC) count and a differential which determines the percentage of WBCs that are lymphocytes. The following calculations can then be made and reported:

Absolute lymphs = WBC count x % lymphs

Absolute CDx = Absolute lymphs x % CDx

COMBINATION     POPULATION IDENTIFIED

CD4+/CDw29+             Helper/effector, more mature memory cells
CD4+/CD45R+             Suppressor inducer, less mature nonmemory cells
CD4+/Leu8+              Suppressor inducer, some helper function
CD4+/Class II MHC       Activated cells, immature cells
CD4+/CD25+              Activated cells (IL2 receptor)
CD4+CD38+               Immature cells, activated cells
CD8+/CD11b+             Of the CD11b+ cells the suppressors are bright CD8+ and NK are dim
                        CD8+
CD8+/CD28+              Cytotoxic precursor/effector cells
CD8+/CD57+              Cytotoxic function
CD8+/Class II MHC+      Activated cells, immature cells
CD8+/CD25+              Activated cells (IL2 receptor)
CD8+/CD38+              Immature cells, activated cells
CD16+/CD57+             Low NK activity
CD16+/CD56+             Most potent NK activity

For acute leukemias, immunophenotyping can be supportive evidence in determining the lineage of the leukemic cell. It is important to note that there is no leukemic specific marker and it is the composite phenotype that must be interpreted. Commonly used markers for leukemias are available.

The acute leukemias are broken into two major groups, acute lymphoblastic leukemia and acute non-lymphocytic leukemia (contains the myeloid leukemias, erthryocytic leukemias, and megakaryocytic leukemias).

Acute Lymphoblastic Leukemia (ALL)

In children under 15 years of age, acute lymphoblastic leukemia (ALL) is the most common malignancy. The incidence of ALL peaks between 3 to 5 years of age, but can be seen in infants under one year of age and in young adults (comprises approximately 20% of adult leukemia cases). The cure rate is more favorable in children between 1 and 10 years of age than in adults. Over half of children with favorable prognostic factors are cured and 85% achieve remission. The morphologic classification is based on the light microscopic appearance which is distinctive with nuclear pleomorphism, 1-2 indistinct nucleoli, usually a high nuclear to cytoplasmic ratio, and coarse nuclear chromatin. The cytochemical stain, myeloperoxidase, is absent.

Most ALL cases are B-cell proliferations (approximately 75-85%) but can very in their degree of maturity. Usually the leukemias arise from very early B-cells. Initial diagnosis relies upon two monoclonal antibodies, CD19 and HLA-DR. The CD19 antigen is the earliest B-cell specific antigen and precedes the expression of HLA-DR (which is not specific to the B-cell lineage). Other monoclonal antibodies can be useful in classifying B-cell leukemia, CD10, CD20, CD21, and CD24, cytoplasmic immunoglobulins, and surface immunoglobulins. CD21, a mature B-cell marker, is generally not expressed in B-cell ALL nor are T-cell antigens. Remember if surface markers of more than one cell line is present the possibility of a mixed or biphenotypic leukemia should be considered.

The most common B-cell ALL phenotype (approximately 50% of cases) expresses CD10 (common acute lymphoblastic leukemia antigen, cALLa), CD19, CD20, terminal deoxynucleotidyl transferase (TdT), and HLA-DR and does not express cytoplasmic and surface immunoglobulin. This subtype of B-cell ALL has the most favorable prognosis. The next most common (approximately 30% of cases) expresses all of the above with the exception of CD20. Rarely, ALL cases express just CD19 alone or CD19, CD20, HLA-DR, CD10, and either surface or cytoplasmic immunoglobulin. TdT can be present in either B or T-cell ALL. HLA-DR can be helpful to differentiate immature from mature B-cells since it has a more intense reaction in immature B-cells.

A leukemic manifestation of Burkitt s lymphoma which is a very aggressive disease is a morphologically distinct form of B-cell ALL. It is characterized by large uniform cells, prominent nucleolus, and moderately abundant deep basophilic cytoplasm which may have small vacuoles. These vacuoles stain Oil Red O positive. These cells have a B- cell immunophenotype with detectable surface immunoglobulin. Burkitt s has the worst prognosis of ALL.

T-cell ALL is seen in approximately 15-25% of cases and tends to be a more aggressive disease. The typical clinical presentation is an older male who has a high blast count in the peripheral blood and mediastinal masses. The diagnosis of T-cell ALL by phenotyping is much more difficult than B-cell ALL for several reasons.

• In T-cell ALL there is no marker for monoclonality such as the immunoglobulin light chain (kappa or lambda) in B-cell ALL.
• There are few markers which are detected only in the early phases of T-cell development and these markers occur uncommonly in T-cell ALL.
• HLA-DR is usually absent in T-cell ALL.
• Benign proliferations can be seen and T-cells are normally prominent in most specimen types (60-80% of the lymphocytes).

The most sensitive antibody is CD7 which is directed to the pan-T antigen. Approximately 95% of T-cell ALL will express CD7 and a diagnosis of T-cell should be examined closely if CD7 is not expressed. However CD7 alone is not a marker specific for T-cell ALL since it has been reported to be aberrantly expressed in B-cell ALL and acute myeloid leukemias. Other useful markers in T-cell ALL are CD1a (except in thymus where thymocytes normally express CD1a), CD2, CD3, and CD5. CD5 and CD2 are expressed in most cases of T-cell ALL.

Acute Non-Lymphocytic Leukemia

Acute non-lymphcytic leukemia (ANLL) is composed of acute myeloid leukemia (AML) and the rarer leukemias acute erythrocytic and acute megakaryocytic leukemia. Immunophenotyping is the most useful in differentiating AML from ALL and has not been particularly useful in subclassifying AML or for prognostic purposes. AML is most commonly seen in adults between the ages of 15 and 40 years of age. Only about 20% of childhood leukemias are AML.

Light scattering properties can be very useful in identifying the blast cells of AML which tend to be found on forward versus orthogonal scatter as well circumscribed population about the location where normal lymphocytes are seen. Since bone marrow is very heterogeneous in nature, the finding of a single-cell population of similar size and characteristics should be very suspicious for a malignancy. An antibody is considered positive if greater than 20% of the cells express the antigen.

Immunophenotypic classification of AML does not very well match the strict morphologic classifications currently used by pathologists. A panel of specific antibodies including CD13, CD14, CD33, and CD34 is used to indicate if the cells are of a myeloid lineage and then whether there is any degree of monocytic lineage involvement. Also antibodies such as HLA-DR and CD45 are included. These antibodies are not myeloid lineage specific, but are generally seen in AML. The lack of expression of HLA-DR found in most acute progranulocytic leukemias (APL), a subtype of AML, is useful in its diagnosis.

In all forms of AML, the demonstration of the lack of of expression of lymphocyte specific markers is helpful by means of exclusion. However, the presence of these markers does not necessarily exclude the possibility of AML or a biphenotypic leukemia. CD7 is found in up to 25% of AML cases.

CD14 is virtually restricted in its expression to mature monocytes. Therefore it is useful to separate monocytes from blasts or monocyte precursors. CD11c along with CD14 expression can be very useful in detecting acute myelmonocytic and acute monocytic leukemias since it is generally not expressed in other forms of AML or ALL.

Chronic lymphocytic leukemia (CLL) is a disease of older adults, 50-70 years of age, and occurs more commonly in males than females (a 2:1 ratio). CLL is commonly discovered on a routine peripheral blood smear. The disease is a clonal expansion of an immunologically competent lymphocytes involving the bone marrow, lymph nodes, and or spleen. The disease is almost indistinguishable from well differentiated lymphoma and is generally referred to as CLL if the bone marrow is extensively involved. If the disease is primarily of the lymph node or spleen then the term well differentaited lymphoma or small lymphocytic lymphoma is used. Morphologically the cells are indistinguishable from normal mature lymphocytes.

The majority of CLL are monoclonal B-cells which have a monotypic expression of either kappa or lambda light chains and react with CD5. The staining intensity of CLL with CD5 is usually greater than either with kappa or lambda light chains. This finding can be helpful in distinguishing CLL from a reactive lymphocytosis.

Prolymphocytic Leukemia

Prolymphocytic leukemia is a more aggressive form of CLL. Like CLL pan-B cell markers are positive (CD19, CD20, CD22, and CD24). The major distinguishing finding is that the monotypic surface immunoglobulin staining is present in high density.

Hairy Cell Leukemia

Hairy cell leukemia (HCL) is a rare leukemia composed of B-cells with a slightly less mature morphology and a distinctive appearance with cytoplasmic projections. HCL reacts with pan-B cell markers (CD19, CD20, CD22, and CD24) but when compared to CLL it has increased surface immunoglobulin staining.

Chronic Myelogenous Leukemia

If the malignancy is derived from a T-cell, it is more difficult to establish the presence of maligancy due to the lack of phenotypic markers of T-cell clonality. Therefore, immunophenotypic evidence of T-cell malignancies rests on abnormal/aberrant antigen expression and or the presence of a DNA abnormality. In summary, to arrive at a diagnosis of lymphoid neoplasm by flow cytometry the case must show 1) immunophenotypic abnormalities and/or 2) abnormal DNA ploidy (reflective of an increase or decrease in chromosome number).

Immunophenotyping in Hodgkin s lymphomas are more useful in ruling out a diagnosis of Hodgkin s disease than in establishing a diagnosis. In general, it is almost impossible to distinguish a Hodgkin s lymphoma from a benign T-cell proliferation. Small lymphocytic lymphomas are maily (98%) of B-cell origin and express the B cell associated markers (CD19, CD22, and CD24). The rare T-cell small lymphocytic lymphomas usually express a mature T-cell helper immunophenotype (expressing CD2, CD3, CD4, CD5, CD7, and not expressing CD8). Follicular lymphomas are of B cell origin and express surface immunoglobulin,most of the pan-B cell markers, and very frequent CD10 may be positive.

Quality Assurance of Immunophenotyping
DNA Cell Cycle Analysis