Discuss the methods that could be used to

separate CD4+T cells from anticoagulante

whole blood.

Introduction:

T-cells (or T-lymphocytes) are white blood cells that play important roles in the immune
system. There are two main types of T-cells. One type has molecules called CD4 on its
surface; these `helper' cells orchestrate the body's response to certain micro-organisms
such as viruses. The other T-cells, which have a molecule called CD8, destroy cells that
are infected and produce antiviral substances.

HIV is able to attach itself to the CD4 molecule, allowing the virus to enter and infect
these cells. Even while a person with HIV feels well and has no symptoms, billions of
CD4 T-cells are infected by HIV and are destroyed each day, and billions more CD4 T-
cells are produced to replace them [1].
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Basic structure of CD+4 cells

CD4 is a co-receptor in the cellular immune response. It increases the avidity of

association between a T cell and an antigen-presenting cell by interacting with non-
polymorphic portions of the complex between class II major histocompatibility complex
(MHC) and T-cell receptor (TCR) molecules, and it contributes directly to signal
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 transduction through its cytoplasmic association with the lymphocyte kinase Lck. CD4
also serves as the high-affinity receptor for cellular attachment and entry of the human
immunodeficiency virus (HIV). The extracellular portion of CD4 comprises four
immunoglobulin-like domains (D1–D4).

Action of CD 4 cells

Structurally CD4 is a 55-kDa glycoprotein with a four immunoglobulin like domain (D1
to D4) which are present at the extracellular surface of the cell, in a hydrophobic
transmembrane region with a long cytoplasm tail having a three serine residues in them
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which have an ability to undergo phosphorylation. Having the help of crystallographic

analysis, it is seen that the D1 domain of the CD4 tend to bind with the 2 domain of
MHC class II molecules. The CD4 molecule also shows the presence of special sequence
of amino acid at its cytoplasmic tail which allows it to interact with 1ck molecule.

Accurate and reliable measures of CD4+ T-lymphocytes (CD4+ T-cells) are essential to
the assessment of the immune system. In a order to separate the CD4+ T cells from the
blood. The blood samples undergo a sequential procedure, where in each step has its own
significance towards it [2, 3].

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 Flow Cytometric anaylysis

One of the most common method for identifying and enumerating the CD4+ T helper
cells (lymphocytes) is flow cytometry. Flow Cytometry is a process in which such
measurements are made while the cells or particles pass, preferably in single file, through
the measuring apparatus in a fluid stream It is an automated system for identifying the
cells based on the scattering of the light as the cell flow through a single passage in the
presence of laser beam. In this process the scattering is read sideways and forward
direction, which determines the cell size and granularity.

Most of the isolated lymphocytes show a uniform appearance. But these cells consists
mainly of functional sub – populations, which can be easily distinguished on the basis of
cell surface protein passed by a particular cell, with the help of specific antibodies. T
cells for example; can be easily subdivided on the basis of the expression of co – receptor
proteins CD4 and CD8.

A flow cytometer is equipped with a device called as fluorescence – activated cell sorter
(FACS) which separates the identified cells possessed with a computer based system. A
pressurize isotonic sheath emerging through a small opening of a flow chamber surrounds
the flow cell and the difference in the pressure between the sheath fluid and sample keeps
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the cells to the center and hence leading to a linear flow of a cells in a single file. This
instrument uses the monoclonal antibodies and cell. The surface protein to determine the
properties of cell subsets. Each cells in the lymphocyte population is tagged with a
particular antibody, labeled with fluorescent dyes or by specific antibodies followed by a
labeled anti immunoglobulin [4].

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 A simplified illustration of Flow Cytometry

During this process when the cells enter the nozzle in the presence of laser beam results
in the scattering of the light (LS) which is detected by optical detectors and is further
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down converted into an electrical impulse through a photo – multiplier tube. These
signals are displayed on the histograms in the form of digital signals. In this the forward
LS represent the cell size and lights at 90 degree represent the internal cell structure
especially the granularity.

Density gradient centrifugation – Ficoll Hypaque

gradient:

Usually the isolation of human lymphocyte from a anticoagulant blood is carried out by
the density centrifugation over a step gradient consisting of a mixture of a carbohydrate
polymer is called ficoll. The density of the ficoll is greater than that of lymphocyte but
less than RBCs and granulocyte. This particular property of ficoll plays a vital role during
the process of isolation.
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 In this process, the anticoagulant blood is carefully layered on the top of the ficoll
solution in the conical centrifuge tube. The centrifugation results in the formation of three
distinct layers as plasma at the top of the tube, a mononuclear layer banding on the top of
the density gradient solution whereas the erythrocytes and the granulocytes at the bottom
of the tube.

Next, a particular lymphocyte population can be isolated from a sample with the help of
cell panning were antibody coated plastic surfaces are used as a binding agent or by
removing unwanted cell by the treatment with specific antibody and a complement to kill
them [5, 7].

The cells can also be separated with the affinity chromatography where the cells are
allowed to pass through a column of antibody coated or nylon coated steel wool resulting
into the differential elution of various populations. These techniques can be used as a pre-
purification step prior to sorting out highly purified populations by FACS.
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Fig 2: Separation of mononuclear cells from the whole blood by Ficoll – Hypaque
centrifugation.

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 Magnetic bead separation:

Magnetic separation is very inexpensive in comparison and many of the tasks for which
flow cytometry is used, such as stem cell, T-cell, and tumor cell isolation can be
performed using magnetic separators. Magnetic cell separation is easily divided into two
classes: using magnetic beads to select cell types, and using the native susceptibility of
cells to select cell types. The use of magnetic beads coated with cell specific antibodies to
separate certain cell types is only about 15 years old, but has blossomed recently as an
affordable way of isolating rare cells. Once the magnetic beads are bound to the cells, a
magnetic field gradient is all that is required to separate them from the bulk. The
magnetic beads, ranging in size from 10 nm to 10 m, are typically a mixture of polymer
and iron oxide particles, Fe2O3 and Fe3O4 [6, 8].
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The most powerful and efficient technique used for isolating CD4+ T helper cells from a
large lymphocyte population is to couple paramagnetic bead to monoclonal antibodies
which are specific to the markers present on the cell surface. The paramagnetic bead
typically consists of the polymer shell with embedded superparamagnetic nanoparticles
of iron oxides. This provides a specific and defined surface for adsorption or coupling of
various bioreactive molecules called as ligands [9].

In this the superparamagnetic shows the magnetic property only when placed within the
magnetic field and show no residual magnetism when removed from the field. The

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 polymer used to cover the bead protects the CD4+ T helper cells from toxic exposure of
iron.
In this technique, the antibody beads specific to CD4+ T helper cells are mixed with the
lymphocytes to be separated and is allowed to pass through the column. As a result the
CD4+ cells are bounded to the beads showing the phenomenon positive selection and the
unbounded cells remain behind [10, 11].

Conclusion:

This is a take note from the discussion that the density gradient centrifugation is the basic
or first step for the separation of the cells present in the whole blood. However the flow
cytometer with with FACS can be used for the purpose of separation of cells in the blood
particularly CD+ 4 cells can be done with a higher percentage of success. It may be
possible that this technique is not effective to separate the cells from a large population.
Therefore the magnetic bead separation which is cost effective and can separate rare cell
also may be better way to perform the experiments.
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Reference:

1) Abbas AK, and Lichtman AH (2003); Cellular and Molecular Immunology,

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 (5th Edition), Saunders, Philadelphia.

2) COCHET Oliver et al; (1998) Immunological Technique Made Easy, John

Wiley And Sons.

3) JANEWAY Jnr. et al; (2001), Immunology, 5th Edition, Garland publication.

4) JOHNSTONE Alan et al;(1996), Immunohistochemistry in practice, 3rd Edition,

Blackwell Science.

5) Weiss, A. & Littman, D. R. Signal transduction by lymphocyte antigen receptors. Cell

76, 263–274 (1994).

6) Lifson, J. D. & Engleman, E. G. Role of CD4 in normal immunity and HIV infection.

Immunol. Rev. 109, 93–117 (1989).

7) S. Miltenyi, W. Mller, W. Weichel, and A. Radbruch, Cytometry 11, 231 (1990).

8) M. Nakamur, K. Decker, J. Chosy, K. Comella, K. Melnik, L. moore, L.

9) C. Lasky, M. Zborowski, and J. J. Chalmers, Biotechnol. Prog. 17, 1145 (2001).

10) H. Taubert, K. Bl¨umke, U. Bilkenroth, A. Meye, A. Kutz, F. Bartel, Lautenschl¨ager,

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E. J. Ulbrich, N. Nass, H. Holzhausen, H. Koelbl, and A. Lebrecht, Gynecol. Oncol.

92, 256 (2004).

11) D. Melville, F. Paul, and S. Roath, Nature 255, 706 (1975).

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