The first step in studying lymphocytes is to isolate them so that their behavior can be analyzed in vitro. Human lymphocytes can be isolated most readily from peripheral blood by density centrifugation over a step gradient consisting of a mixture of the carbohydrate polymer Ficoll-Hypaque™ and the dense iodine-containing compound metrizamide. A step gradient is made by preparing a solution of Ficoll-Hypaque at a precise density (1.077 g/liter for human cells) and placing a layer of this solution at the bottom of a centrifuge tube. A sample of heparinized blood mixed with saline (heparin prevents clotting) is carefully layered on top of the Ficoll-Hypaque solution. Following centrifugation for about 30 minutes, the components of the blood have separated based on their densities. The upper layer contains the blood plasma and platelets, which remain in the top layer during the short centrifugation. Red blood cells and granulocytes have a higher density than the Ficoll-Hypaque solution and collect at the bottom of the tube. The resulting population, called peripheral blood mononuclear cells (PBMCs), collects at the interface between the blood and the Ficoll-Hypaque layers and consists mainly of lymphocytes and monocytes (Fig. A.18). Although this population is readily accessible, it is not necessarily representative of the lymphoid system, because only recirculating lymphocytes can be isolated from blood.
The ‘normal’ ranges in the numbers of the different types of white blood cells in blood, along with the normal ranges in concentrations of the various antibody classes, are given in Fig. A.19.
A-17 Isolation of lymphocytes from tissues other than blood.
In experimental animals, and occasionally in humans, lymphocytes are isolated from lymphoid organs, such as spleen, thymus, bone marrow, lymph nodes, or mucosal-associated lymphoid tissues—in humans, most commonly the palatine tonsils (see Fig. 12.6). A specialized population of lymphocytes resides in surface epithelia; these cells are isolated by fractionating the
Diluted anticoagulated blood (left panel) is layered over Ficoll-Hypaque™ and centrifuged. Red blood cells and polymorphonuclear leukocytes or granulocytes are denser and travel through the Ficoll-Hypaque™, while mononuclear cells consisting of lymphocytes together with some monocytes band over it and can be recovered at the interface (right panel).
Fig. A.18 Peripheral blood mononuclear cells can be isolated from whole blood by Ficoll-Hypaque™ centrifugation.
centrifugation diluted blood Ficoll-HypaqueTM (specifc gravity =1.078)
Fig. A.19 The major cellular and humoral components of human blood. Human blood contains B cells, T cells, and myeloid cells, as well as high concentrations of antibodies and complement proteins.
epithelial layer after its detachment from the basement membrane. Finally, in situations where local immune responses are prominent, lymphocytes can be isolated from the site of the response itself. For example, in order to study the autoimmune reaction that is thought to be responsible for rheumatoid arthritis, an inflammatory response in joints, lymphocytes are isolated from the fluid aspirated from the inflamed joint space.