FLOW CYTOMERY

Alan Winkelstein, Medical Director

INTRODUCTION

Flow cytometry is an important adjunct in the diagnosis and management of an increasing number of clinical disorders.  It serves to both identify and enumerate specific types of cells, a process referred to as immunophenotyping.  The principles underlying flow cytometry are comparatively simple.  Cell suspensions (blood, bone marrow, or suspensions of tissue cells) are incubated with antibodies to a unique cell membrane component.  For example, T-helper/inducer lymphocytes are characterized by the presence of the membrane associated CD4 molecule; other lymphoid cells do not possess this specific protein.  Thus, an antibody to the CD4 molecule selectively reacts with this lymphoid subset.  The term “CD” indicates “Cluster of Differentiation” and is the accepted nomenclature for cell membrane-associated antigens.

A flow cytometer is the analytic instrument used to automatically distinguish between labeled and unlabeled cells.  In order to recognize a cell-bound antibody, the latter is coupled to a fluorochrome, a visible dye, such as fluorescein or phycoerythrin.  Analysis is performed by passing the cells through a narrow channel that intersects a laser light beam.  The wave length of the emitted light allows appropriate light sensors to recognize cells bearing the fluorochrome-labeled antibody.  These sensors are connected to a computer, which records the dye’s staining intensity of each cell.

CLINICAL USES

A.     HIV Infection

The most widely used application of flow cytometry is in the management of HIV-infected patients.  The causative virus selectively infects and destroys T-helper/inducer (CD4) lymphocytes; this leads to a progressive impairment in the immune defense system.  The resulting immunodeficiency predisposes to both opportunistic infections (such as Pneumocystitis Carinii Pneumonia) and to certain malignancies.  The importance of these cells is illustrated by the CDC’s recent decision to broaden its definition of AIDS to include HIV-infected individuals, regardless of symptoms, who have CD4 cell counts <200/mm³. 

Clinically, most HIV-infected patients experience a long asymptomatic phase; the median duration is approximately 10 years.  However, this phase is characterized by a progressive loss of CD4+ cells.  Until recently, it was believed that prophylactic treatment with the antiretroviral agent Zidovudine (AZT) could prolong the asymptomatic phase of this illness.  Therapy was recommended when the CD4 count fell below 500 cells/mm³.  However, recent studies have cast doubt on the benefits of this prophylactic therapy.  Current recommendations are that decisions about prophylactic antiretroviral therapy should be individualized.

By contrast, anti-retroviral therapy is clearly indicated in symptomatic patients.  Another milestone useful in following HIV-infected patients is a CD4+ cell concentration <200/mm³.  Below this level, patients should be considered candidates for pheumocystitis prophylaxis.  Recent recommendations suggest a specific panel of antibodies in which the actual numbers of CD4+ counts should be obtained at three to six month intervals.

Quantitation of lymphocyte subsets can also be useful in evaluating patients suspected of having a congenital or acquired immune deficiency.  This analysis is generally recommended as part of the specific evaluation of any patient who appears to be unduly susceptible to infections.

B.      Acute Leukemia

A second major use for flow cytometry is in the classification of acute leukemia.  In most cases, immunophenotyping can distinguish between lymphoblastic (ALL) and myeloblastic (AML) forms.  This distinction is frequently difficult by routine morphological criteria and the two forms require different therapies and have different prognoses.  In patients with AML, phenotyping can also be useful in detecting certain relatively uncommon subsets, such as acute megakaryocytic leukemia (M7) and acute erythroleukemia (M6).  Current data also suggest that the presence of certain antigens, such as CD34, may imply a poorer prognosis.  Unfortunately, there is relatively poor correlation between the FAB morphological classification of AML and leukemia phenotyping.

Among ALL, immunophenotyping can subclassify these malignancies into those due to the clonal transformation of T cells, B cells, or pre-B cells.  Pre-B cell ALL is the most prevalent form of ALL and has the best prognosis.  Acute T cell ALL accounts for 15 to 25% of the cases; it typically occurs in older children and adults.  B lymphocytes bearing either kappa or lambda light chains.

Flow cytometry is able to distinguish specific types of lymphoproliferative neoplasms.  One of the best categorized is B-CLL.  In this disease, the malignant cells are characterized by the co-expression of both pan B cell antigens and a single T cell antigen, CD5.  In a patient with persistent lymphocytosis, the coexpression of B cell antigens and CD5 distinguishes CLL from other leukemias due to the neoplastic transformation of lymphocytes, the leukemic phases of a non-Hodgkin’s lyumphoma and reactive lymphocytosis.  Recent studies suggest that CD5+ B cells may have important immunoregulatory functions and that these cells may be intimately related to the impaired humoral immunity that complicates B-CLL.

Likewise, other antigens, such as CD11c, which is strongly expressed in Hairy Cell Leukemia, can help in establishing the diagnosis of specific B cell neoplasms.  Immunophenotyping can distinguish specific types of chronic T cell lymphorproliferative diseases, such as large granular cell leukemia (LGL).  This neoplasm is being recognized with increasing frequency and is known to be associated with several complications including severe neutropenia and pure red cell aplasia.  For unexplained reasons, patients with LGL have a high incidence of co-existing rheumatoid arthritis. 

C.      Stem Cell Enumeration

Another growing use of flow cytometry is in the quantitation of stem cells for marrow reconstitution following myeloablative therapy.  Analysis of either bone marrow or peripheral stem cell harvests for the number of CD34+ cells appears to be a useful measure of stem cell content.  Evidence is accruing that the success and rapidity of engraftment can be predicted using this flow cytometric-based measurement.

Copies of the Transfusion Medicine Update can be obtained by contacting Deborah Small at (412) 209-7320 or
by e-mail:  dsmall@itxm.org.