CELLULAR IMMUNITY Specific cellular immunity is mediated by T cells, and defects affecting these lymphocytes underlie the most severe immune deficiencies. Because antibody production requires intact T cell function, most T cell defects lead to combined (cellular and humoral) immune deficiency. Any child presenting with recurrent or severe viral and/or bacterial illnesses or opportunistic infections in the first year of life requires evaluation of T cell number and function. Similar considerations apply to adults with these types of infections. In the pediatric age group, most cases are due to congenital immune deficiency. In older children and adults, the major causes are HIV infection and iatrogenic immune suppression due to therapy for autoimmune disease, malignancy, or transplantation. Occasionally, mild forms of primary combined immune deficiency escape diagnosis until adulthood.
Complete blood count with differential and blood smear The complete blood count (CBC) with differential and blood smear must be performed in all cases. Normal ranges for cell populations in adults are well established, and the normal ranges for lymphocytes in children are also available [3,4]. In many diseases, cell populations are initially normal, and then decline over time. Thus, normal results in the past cannot be relied upon as a reflection of the current state.
The CBC establishes the presence or absence of lymphopenia, and any associated gross hematologic abnormalities, some of which may greatly assist in diagnosis. Sequential CBCs can also be used to monitor the courses of various disorders. However, a single finding of lymphopenia should be interpreted with caution, since transient lymphopenia is frequently found in a variety of common infectious illnesses [5].
On the other hand, significant lymphopenia should not be ignored, since lymphopenia may be the first indication of SCID (Severe Combined Immune Deficiency [6]. It is also critical to note that normal lymphocyte counts in infants are much higher than in older children and adults.
In an infant with any significant infection and a total lymphocyte count <3,000 cells/cubic mm, flow cytometry (see below) should be performed. In the absence of other indicators of immune dysfunction, there should at the very least be subsequent measurement of the lymphocyte count to document normalization. Persistent lymphopenia requires further investigation of immune function.
Other abnormalities of the complete blood count and differential are discussed separately.
Determination of lymphocyte subpopulations by flow cytometry Flow cytometry is appropriate in the initial evaluation of all patients with opportunistic infections or severe or persistent lymphopenia [7]. Standard flow cytometry analysis will be abnormal in all cases of severe combined immune deficiency (SCID), and in many instances of other combined immunodeficiencies.
Opportunistic infections may not be evident among patients with milder variants of immune deficiency syndromes, early in the course of severe combined immune deficiency (SCID), and in many instances of other combined immunodeficiencies. Such children frequently present with recurrent bacterial sinopulmonary disease or severe cases of common childhood infections; similar considerations apply to adults. Most of these patients are initially screened with a CBC, measurement of serum immunoglobulins and specific antibody responses, and possibly cutaneous delayed hypersensitivity testing (see below). When these tests are not diagnostic of a particular syndrome or when marked abnormalities are present, analysis of lymphocyte subsets by flow cytometry should be undertaken.
Method For analysis by flow cytometry, blood cells are mixed with fluorochrome-labeled monoclonal antibodies that bind to cell surface markers that distinguish various functional categories of lymphocytes. The flow cytometer detects fluorescence and counts the number of cells that bind the monoclonal antibody on their surface. Results are expressed as a percentage of cells analyzed.
It is imperative that either a CBC and differential be performed on a blood specimen obtained at the time of the flow cytometric analysis, or the cytometer itself be used to determine the lymphocyte number. This analysis permits the calculation of the absolute numbers of each lymphocyte subset. It is possible for the percentage of a particular subset to be abnormal while the total number of cells is within the normal range, and vice-versa. An absolute deficiency, rather than a relative (percentage) deficiency, is of much greater clinical significance.
The fundamental set of markers commonly used and the lymphocyte populations they define are below. The nature and derivation of the nomenclature of the markers is beyond the scope of this review, and may be found in any basic immunology text.
Markers commonly used for assessment of lymphocyte subsets by flow cytometry
Marker name (Cell type) Comment
CD3 (T cells) Expressed on all T cells and no other cell type
CD4 (T cell subset) Predominantly helper/inducer T cells
CD8 (T cell subset) Predominantly cytotoxic T cells; expressed by up to one-third of natural killer cells
CD19 or CD20 (B cells)
CD16 (Natural killer cells) Some NK cells may not express CD16
CD56 (Natural killer cells) Expressed on the majority of NK cells
CD57 (Natural killer cells) Expressed on the majority of NK cells; combinations of CD16, CD56 and CD57 will more reliably evaluate NK cell number than any marker alone
CD45 (All leukocytes) Positive control
CD14 (Monocytes) Negative control
Thought this might be useful information.... it is from a medical reference available to most physicians. It was last updated in June 2009 with a literature review in September 2009.
Hopefully, I'll be abel to provide further detail! ~ JT
