Cells found in blood
Blood cells
White blood cells Red blood cells Platelets
(Leucocytes) (Erythrocytes) (Thrombocytes)
Human blood contains many cell types of variable size and internal complexity. The three basic cell types are red blood cells (erythrocytes), white blood cells (leucocytes)
and platelets (thrombocytes). White blood cells can be further subdivided as shown next.
Cells found in blood
White blood cells
(Leucocytes)
Granulocytes Monocytes Lymphocytes
White blood cells (leucocytes) are further grouped into monocytes, lymphocytes and granulocytes. As shown in the next three slides, all of these groups also have cell subsets.
Cells found in blood
Granulocytes
Neutrophil Basophil Eosinophil
Granulocytes are composed of neutrophils, basophils and eosinophils.
Cells found in blood
Monocytes
Dendritic cell Macrophage
Monocytes can be dendritic cells or macrophages.
Cells found in blood
Lymphocytes
T Lymphocyte B Lymphocyte Natural Killer cell
Lymphocytes are composed of T lymphocytes, B lymphocytes and Natural Killer cells. T lymphocytes can be further subdivided as shown next.
Cells found in blood
T Lymphocytes
CD4+ Helper CD8+ Cytotoxic
T Lymphocyte T Lymphocyte
T lymphocytes can either be CD4+ T helper lymphocytes or CD8+ cytotoxic T lymphocytes.
Summary of cells found in blood
Leucocytes Erythrocytes
Granulocytes
Monocytes
neutrophil
basophil
dendritic cell
macrophage
Lymphocytes Platelets
CD8+ eosinophil
Cytotoxic
T lymphocyte
CD4+ Natural
Helper Killer
T lymphocyte Cell
B lymphocyte
Human blood contains many cell types of variable size and internal complexity. The three basic cell types are red blood cells (erythrocytes), white blood cells (leucocytes)
and platelets (thrombocytes). White blood cells are further grouped into monocytes (macrophages and dendritic cells), lymphocytes (T lymphocytes, B lymphocytes and
Natural Killer cells) and granulocytes (neutrophils, basophils and eosinophils). T lymphocytes can be subdivided further into CD4+ Helper T lymphocytes and CD8+ Cytotoxic
T lymphocytes.
Absolute CD4 count
Lymphocytes
CD4+ Helper CD8+ Cytotoxic B Lymphocyte Natural Killer cell
T Lymphocyte T Lymphocyte
An absolute CD4 count is the number of CD4+ Helper T lymphocytes present in 1ml of blood. These cells carry the CD4 cell surface molecule.
CD4 percentage
Lymphocytes
CD4+ Helper CD8+ Cytotoxic B Lymphocyte Natural Killer cell
T Lymphocyte T Lymphocyte
The CD4 percentage is expressed as the ratio of CD4+ Helper T lymphocytes to the total population of lymphocytes in blood. In children where lymphocytes are present in
higher numbers, the absolute CD4 count is less reliable as a marker of HIV disease state and the CD4 percentage is preferred.
(1) Flow cytometry using light scatter
1 Sample 2 Sheath
Sheath 5 granulocytes
fluid
Forward
Side scatter (SS)
scatter
detector
monocytes
Flow
chamber FS
Side
3 scatter 4 lymphocytes
Photon detector Forward scatter (FS)
beam Lens
SS
mirror
Laser
Flow cytometry is a method used to differentiate and count cells in a sample. (1) A sample containing cells is transferred to a tube (2) that separates the cells from each
other. (3) The light properties of individual cells are measured as they pass through a photon beam generated by a laser. (4) Light transmitted at 180° is recorded as forward
scatter (FS) and light reflected at 90° is recorded as side scatter (SS). (5) A graphical plot of SS versus FS depicts the relative size and internal complexity of each cell and
this information can be used to quantify particular cell subsets.
(2) Flow cytometry using fluorescence
1 2 Sheath
Sample CD4+ CD3+
Sheath
fluid 5 lymphocytes
CD4
Flow
chamber
Photon
beam
3 Lens CD3
FL1
4
FL2
FL3
mirror
Laser Fluorescent
light
detectors
FL4
Flow cytometry is also used to differentiate and count cells labelled with fluorescent dyes. (1) Antibodies that bind cell surface molecules are added to the sample. Different
antibodies are labelled with different colours. (2) A sample containing cells with bound antibody is transferred to a tube that separates the cells from each other. (3) Fluorescent
light emitted from individual cells is measured as they pass through the photon beam. (4) Various filters and light detectors record the fluorescence signals. (5) Fluorescence
intensity can be plotted to show the subset of cells binding the label.
(1) CD4+ Helper T lymphocyte counting by CD3/CD4 staining
3
flow
cytometer
anti-CD4
antibody 4
anti-CD3
antibody
2
sample
1 CD4+ CD3+
lymphocytes 5
CD4
CD3
This method uses antibodies that bind the CD3 and CD4 cell surface molecules. The antibodies are coupled to different fluorescent dyes. (1) A fresh blood sample is provided.
(2) The antibodies are added and allowed to bind the cell surface molecules. (3) A sample is transferred to the flow cytometer. (4) The fluorescent signals are recorded. (5)
The fluorescence signals are plotted to obtain a gate depicting the CD3/CD4 double positive cells. The percentage is calculated by the instrument and the absolute CD4
count inferred by single or double-platform methods.
CD4+ Helper T lymphocyte counting by CD3/CD4 staining
CD4+
Helper
T lymphocyte
fluoresecent CD4+ CD3+
dye (red) lymphocytes
anti-CD4 fluoresecent
A
CD4
CD4 antibody dye (green)
CD45
TCR
CD3
complex
anti-CD3
α β
}
antibody CD3
CD4+
δ ε ε γ ζ ζ Helper
T lymphocyte Cell
membrane
Calculating the number of CD4+ Helper T lymphocytes/ml of blood with CD3/CD4 staining uses the percentage of CD3+/CD4+ cells counted by flow cytometry (box A). The
dual-platform method requires a haemocytometer to count the total leucocytes (WCC) and the lymphocyte differential (LD). The absolute CD4+ Helper T lymphocyte count
is calculated as the CD3+/CD4+ cell percentage of the total lymphocytes (LD x WCC). The single-platform method uses control beads of known concentration or a volumetric
flow cytometer that measures blood volume as a calibration standard.
Blood cells labelled by CD3/CD4 staining
Leucocytes Erythrocytes
Granulocytes
Monocytes
neutrophil
basophil
dendritic cell
macrophage
Lymphocytes Platelets
CD8+ eosinophil
Cytotoxic
T lymphocyte
CD4+ Natural
Helper Killer
T lymphocyte Cell
B lymphocyte
HIV infection is characterised by a loss of CD4+ Helper T lymphocytes. An absolute count of these cells in blood is a useful marker of disease state. Flow cytometry is a
method used to measure the number of these cells in blood. A common strategy to identify these cells uses fluorescent dyes conjugated to antibodies against CD3 and CD4
cell surface markers. CD3 is expressed on CD4+ Helper T lymphocytes and CD8+ Cytotoxic T lymphocytes. CD4 is expressed on macrophages and CD4+ Helper T
lymphocytes. CD3/CD4 double-positive cells are the CD4+ Helper T lymphocytes.
(2) CD4+ Helper T lymphocyte counting by CD45/CD4 staining (Panleucogating)
3
flow
cytometer
anti-CD45
antibody 4
anti-CD4
antibody
2
sample
1 CD45+
leucocytes 5
CD4+
lymphocytes
A
Side scatter
Side scatter
B
CD45 CD4
This method uses antibodies that bind the CD45 and CD4 cell surface molecules. The antibodies are coupled to different fluorescent dyes. (1) A fresh blood sample is
provided. (2) The antibodies are added and allowed to bind the cell surface molecules. (3) A sample is transferred to the flow cytometer. (4) The fluorescent signals are
recorded. (5) The fluorescence signals and side scatter are plotted to obtain gates depicting the CD45 and CD4 positive cells. The percentages are calculated by the
instrument and the absolute CD4 count inferred by single or double-platform methods.
CD4+ Helper T lymphocyte counting by CD45/CD4 staining (Panleucogating)
CD45+
leucocytes
CD4+
Helper
T lymphocyte A
Side scatter
fluoresecent
dye (green) fluoresecent
anti-CD45 dye (red)
antibody
anti-CD4
antibody CD45
CD45 CD4
TCR
CD3 CD4+
complex lymphocytes
α β
}
CD4+
Helper
δε ε γ ζζ
Side scatter
T lymphocyte
Cell B
membrane
CD4
Calculating the absolute number of CD4+ Helper T lymphocytes/ml of blood with CD45/CD4 staining uses the ratio of CD4+ lymphocytes to CD45+ leucocytes. The dual-
platform method requires a haemocytometer to count the total leucocytes (WCC). The absolute CD4+ Helper T lymphocyte count is calculated as the ratio of CD4+ lymphocytes
(box B) to total CD45+ leucocytes (box A) applied to the WCC. The single-platform method uses labelled control beads of known concentration or a volumetric flow cytometer
that measures blood volume as a calibration standard.
Blood cells labelled by CD45/CD4 staining
Leucocytes Erythrocytes
Granulocytes
Monocytes
neutrophil
basophil
dendritic cell
macrophage
Lymphocytes Platelets
CD8+ eosinophil
Cytotoxic
T lymphocyte
CD4+
Helper Natural
T lymphocyte Killer
Cell
B lymphocyte
HIV infection is characterised by a loss of CD4+ Helper T lymphocytes. A count of these cells in blood is a useful marker of disease state. Flow cytometry is a method used
to measure the number of these cells in blood. An alternative strategy to CD3/CD4 staining to identify these cells uses fluorescent dyes coupled to antibodies against CD45
and CD4 cell surface markers (panleucogating). CD45 is expressed on all leucocytes while CD4 is expressed on macrophages and CD4+ Helper T lymphocytes. Side scatter
is used to distinguish macrophages from Helper T lymphocytes.