Cell Surface Immunophenotyping

Cell Surface Immunophenotyping is a test used to detect the progressive depletion of CD4 T lymphocytes, which is associated with an increased likelihood of clinical complications from acquired immunodeficiency syndrome (AIDS). Test results can indicate if a patient with AIDS is at risk for developing opportunistic infections. It is also used to confirm the diagnosis of acute myelocytic leukemia (AML) and to differentiate AML from acute lymphocytic leukemia (ALL).

All lymphocytes originate from reticulum cells in the bone marrow. Normal hematopoietic cells undergo changes in expression of cell surface markers as they mature from stem cells into cells of a committed lineage. Monoclonal antibodies have been developed that react with lymphoid and myeloid glycoprotein antigens on the cell surface of peripheral blood cells. One kind of lymphocyte that matures in the bone marrow is called a B lymphocyte. B lymphocytes provide humoral immunity (produce antibodies). A second type of lymphocyte matures in the thymus and is called a T lymphocyte. T lymphocytes are responsible for cellular immunity. Finally, there is a group of lymphocytes that has neither T nor B markers. These are called “natural killer cells” and will chemically attack foreign or cancer cells without prior sensitization. Monoclonal antibodies against cell-surface markers are used to identify the various forms of lymphocytes. The absolute numbers and percentages are then counted using flow cytometry. This can be performed on blood or on cell suspensions of tissue.


CD4 helper cells and CD8 cells are examples of T-lymphocytes. T-lymphocytes, and especially CD4 counts, when combined with HIV RNA viral load testing (p. 316), are used to determine the time to initiate antiviral therapy. They also can be used to monitor antiviral therapy. Successful antiviral therapy is associated with an increase in CD4 counts. Worsening of disease or unsuccessful therapy is associated with decreasing T-lymphocyte counts.


There are three related measurements of CD4 T lymphocytes. The first measurement is the total CD4-cell count. This is measured in whole blood and is the product of the WBC count, the lymphocyte differential count, and the percentage of lymphocytes that are CD4 T cells. The second measurement, the CD4 percentage, is a more accurate prognostic marker. It measures the percentage of CD4 lymphocytes in the whole blood sample by combining immunophenotyping with flow cytometry. This procedure relies on detecting specific antigenic determinants on the surface of the CD4 lymphocyte by antigen-specific monoclonal antibodies labeled with a fluorescent dye. The third prognostic marker, which is also more reliable than the total CD4 count, is the ratio of CD4 (T-helper) cells to CD8 (T-suppressor) cells.


Of the three T-cell measurements, the total CD4 count is the most variable. There is substantial diurnal variation in this count. Because it is a calculated measurement, the combination of possible laboratory error and personal fluctuation can result in wide variations in test results. With the CD4 percentage and CD4/CD8 ratios, very little diurnal variation and laboratory error exist. The Multicenter AIDS Cohort Study suggests that the latter two measurements are more accurate than the total CD4 count. However, because the total CD4-cell count was originally thought to be the best marker, this test was used in many of the studies that now form the basis for practice recommendations. It will take time before the more accurate measurements find clinical pertinence in practice recommendations.


The pathogenesis of AIDS is largely attributed to a decrease in the T lymphocyte that bears the CD4 receptor. Progressive depletion of CD4 T lymphocytes is associated with an increased likelihood of clinical complications from AIDS. Therefore CD4 measurement is a prognostic marker that can indicate whether a patient infected with human immune deficiency virus (HIV) is at risk for developing opportunistic infections. The measurement of CD4-cell levels is used to decide whether to initiate Pneumocystis jiroveci pneumonia prophylaxis and antiviral therapy and for determining the prognosis of patients with HIV infection.


Both immunodeficiency and the dosage of immunosuppressive medications used after organ transplant are also monitored with the use of this cell surface immunophenotyping. Lymphomas and other lymphoproliferative diseases are now classified and treated according to the predominant lymphocyte type identified. In some instances, the prognosis of these diseases depends on this lymphocyte phenotyping.


The U.S. Public Health Service has recommended that CD4 prognostic markers be monitored every 3 to 6 months in all persons infected with HIV. Because the CD4 counts gradually fall in virtually all such patients, periodic review of the count can be emotionally stressful for both the patient and physician. The patient confronts his or her mortality as the health care provider confronts his or her ultimate powerlessness against the relentlessly advancing infections.


As the CD4-cell measurements decrease, the probability of developing AIDS increases. Forty-eight percent of patients can be expected to develop AIDS within 6 months when their CD4 count is less than 100 cells/mm3. It is recommended that antiviral therapy be started in patients whose CD4 count is less than 500 to 600 cells/mm3P. jiroveci pneumonia prophylaxis should be started when the CD4 count is less than 200 to 300 cells/mm3.


CD4 prognostic markers also can be useful in guiding the approach to the patient’s symptoms. Complaints such as cough and headache are common in most people; however, in patients infected with HIV, these symptoms often raise concerns about opportunistic infections. If the CD4 cell count exceeded 500 cells/mm3 in the past 6 months, there is a very low probability that these symptoms result from opportunistic infections. Knowing this, the patient and physician can feel comfortable with routine care.


Flow cytometry is able to analyze thousands of cells in less than a minute. The flow cytometer has three components in testing: an optical, a fluid, and an electronic system. The optical system consists of an argon laser that emits a single wavelength of light at 488 nm (blue region). Cells are labeled with one of several fluorochromes, including fluorescein (FITC), phycoerythrin (PE), and peridinin-chlorophyll protein (PCP), as a result of monoclonal antibody–fluorochrome conjugate binding to a specific blood cell. The fluorochromes are excited by the laser and emit green (FITC), orange (PE), and red (PCP) light that is measured through optical filters designed to capture their specific wavelength. The fluid system introduces the fluorochrome-bound cells in suspension into a pressurized sheath of fluid that travels through a clear cuvette. The laser light intersects a stream of cells that pass single file through the cuvette. The electronic system measures electronic signals from the detectors that provide measures of the magnitude of fluorescence intensity and the extent of light scatter associated with each cell as it passes through the laser. Most clinical flow cytometers measure five parameters on each cell: two nonfluorescence measures (magnitude of forward and side scatter) and three fluorescence measures (green, orange, and red light intensity). Multiple markers can be used simultaneously to identify different cell populations.


By using a combination of monoclonal antibodies recognizing B-cell, T-cell, and myeloid antigens, it is possible to confirm the diagnosis of acute myelocytic leukemia (AML) and to differentiate AML from acute lymphocytic leukemia (ALL) if morphology and traditional immunohistochemistry are inconclusive (<15% of cases). It is also helpful in identifying mixed patterns of leukemia that may affect prognosis and treatment. Furthermore, flow cytometric cell surface immunophenotyping is extremely helpful in differentiating various forms of immunodeficiency diseases.



Normal Levels

T cells:                                      60-95%   and  800-2500 Cells/μL

T-helper (CD4) cells:           60-75%   and   600-1500 Cells/μL

T-suppressor (CD8) cells: 25-30%   and   300-1000 Cells/μL

B cells:                                        4-25%    and    100-450  Cells/μL

Natural killer cells:              4-30%    and    75-500 Cells/μL


  • The Normal ratio between T-helper cells and T-suppressor cells should exceed 1. In other words the T-helper cells count must exceed the T-suppressor cells count.
  • The lymphocytes count normally increases slightly during the late morning hours. This slight increase may affect the results of the Cell Surface Immunophenotyping for patients who has low levels.



Causes of High Levels

Chronic Lymphocytic Leukemia is accompanied with an increase of lymphocytes.


B-cell Lymphoma is expected to increase the B-lymphocyte counts in the tumor tissues or in the peripheral blood.


T-cell Lymphoma is expected to increase the  T-lymphocyte counts in the tumor tissues or in the peripheral blood in conditions where the bone marrow is heavily affected by the tumor.


Steroids and some steroid based treatments may increase the lymphocyte count.


Viral disorders that cause High Lymphocyte Count (Lymphocytosis) can also lead to high levels.




Causes of Low Levels:

– Human Immunity Virus (HIV) causes the T-Helper cells count to be below 200 cells/μL in late stages when the patient starts to show symptoms for Acquired Immune Deficiency Syndrome  (AIDS).


DiGeorge Syndrome with Thymic Hypoplasia causes the B-Cell Lymphocyte count to be less than normal due to genetic code deletion in Chromosome 22 which causes the body to either produce very few amounts of B-Cell Lymphocytes or not to produce them at all.


Organ Transplant causes a reduction of lymphocyte count which is also an appreciated result because immunosuppression is required to prevent the body from attaching the new organ.


Immunosuppressive Drugs decreases the count of all types of White Blood Cells including the Lymphocytes.


– Recent viral infections may lower the T-lymphocyte counts.


– Cause of Low Lymphocyte Count such like Radiation Thereby and treatment with Alkylating agents also cause low levels to occur.