Investigation of Lymphocytosis
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Introduction
Lymphocytosis or increased blood lymphocyte count is common and occurs at some time in most people, usually in association with viral infections. The major causes of lymphocytosis are listed in Table 1. Lymphocytosis is commonly short lived, and investigation of such cases by immunophenotyping is usually unrewarding. Uncommonly. lymphocytosis may indicate a lymphoproliferative disorder. This is more likely if one or more of the features listed in Table 2 is present, in which case investigation by immunophenotyping is warranted..
Analysis of Lymphocyte Subsets
The first step in the investigation of lymphocytosis is to assess the major lymphocyte subsets: T cells, B cells and NK cells. The T cells are further divided into CD4 T cells (helper cells) and CD8 T cells (cytotoxic cells). An anticoagulated blood sample is required, and the preferred anticoagulant is acid-citrate-dextrose (ACD). The test should be performed within 24 hours of collection, because cells may deteriorate on longer storage. In the laboratory, small quantities of blood are added to tubes containing different antibodies labelled with fluorescent dyes. The tubes are then run on a flow cytometer, and the proportions of cells stained with the various antibodies are determined. In a healthy person, roughly 70-80% of lymphocytes are T cells, and approximately equal proportions of the remainder are B cells and natural killer (NK) cells. Of the T cells, the ratio of CD4 to CD8 cells is normally about 2:1. A full blood count tube must be collected at the same time, to determine the absolute lymphocyte count. The absolute counts of the various subsets are then calculated, and these values are usually more informative than the percentages. In children up to 6 years of age, the absolute lymphocyte count is significantly higher than in adults, and the absolute counts of the various subsets are also higher. .
Infections
In viral infection, the elevation in total lymphocyte count is caused by an increase in the absolute count of CD8 T cells. This occurs most dramatically in Epstein-Barr virus infection, but is also associated with other viruses such as acute CMV infection. The cytotoxic CD8 cells are able to kill infected cells, thereby limiting the dissemination of the virus. The CD8 count may remain markedly elevated for many months after the patient recovers. Other infectious diseases that may cause lymphocytosis include toxoplasmosis, in which CD8 T cells are selectively increased, and pertussis, in which all subsets are increased. However, in many infections the blood lymphocyte count remains within normal limits.
Disorders of the Spleen
Patients who have undergone splenectomy, or whose splenic function is otherwise reduced, may have a lymphocytosis. All lymphocyte subsets may be increased, especially NK cells, and the lymphocytosis itself is not clinically significant.
Lymphoproliferative disorders
The most serious cause of lymphocytosis is a lymphoproliferative disorder. If the clinical notes indicate that such a disease is suspected, and immunophenotypic analysis is requested, the laboratory tests the blood sample with a large panel of antibodies, to determine whether the lymphocytes have abnormal markers, and whether there is evidence of monoclonality.
i. B cell lymphoproliferative disorders
All mature B cells express either kappa or lambda antibody light chains. In a normal individual, there is a mixture of kappa and lambda B cells. In a neoplastic disorder, with a monoclonal population derived from a single B cell, there is a predominance of either kappa or lambda cells. The test for kappa and lambda is simple and reliable.
B cell chronic lymphocytic leukaemia (B-CLL) is by far the most common lymphoproliferative disorder associated with lymphocytosis. B-CLL is usually slowly progressive, and in many patients, abnormal cells can be detected in the blood well before the development of symptoms and signs. It is increasingly common for B-CLL to be first suspected when an incidental lymphocytosis is noted in a full blood count. A minority of cases of B cell lymphoproliferative disorder with lymphocytosis are B cell non-Hodgkin lymphoma (B-NHL). (In this condition, even if the total lymphocyte count is not elevated, the abnormal cells can sometimes be detected in the peripheral blood.)
Once a monoclonal B cell population has been identified by kappa and lambda analysis, various other markers are used to distinguish between B-CLL and B-NHL (Table 3). In patients with a lymphocytosis, if the phenotype is consistent with B-CLL, a diagnosis of B-CLL can be made without further investigations, provided the blood film morphology and clinical features are consistent. Immunophenotyping can also confidently identify B-NHL cells, and provide initial clues to classification. However, there are many different subtypes of B-NHL, and full diagnosis depends on a tissue biopsy.
ii. T cell and NK cell lymphoproliferative disorders
CD4 T cell, CD8 T cell and NK lymphoproliferative disorders are much less common than their B cell counterparts, and classification is less satisfactory. It may be difficult to distinguish a reactive lymphocytosis from a lymphoproliferative disorder. If an elevated T cell count is detected as an isolated finding in an otherwise healthy individual, the lymphocyte subset determination should be repeated every 3-6 months. If the condition is post-infectious, the T cell counts are likely to fall gradually; increasing counts favour the possibility of a lymphoproliferative disorder. Immunophenotyping can detect monoclonality in about half the cases of CD4 or CD8 T cell lymphoproliferative disorders by specialized methods.
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