Patrizia Iseli
Dr. Simone Zimmerli, Merck Serono
Comparison of different techniques to charaterize the cellular
composition of blood, lymph nodes and spleen in healty mice
Introduction
Blood consists of red blood cells, white blood cells and platelets. The white blood cells can
be subdivided into lymphocytes, monocytes, neutrophils, eosinophils and basophils. In this
study we focused on the total white blood cell count (WBC), lymphocytes and monocytes.
Lymphocytes are white blood cells that are able to recognize and target pathogenic
microorganisms. They are divided in B-lymphocytes secreting antibodies and T-lymphocytes
which are able to destroy infected cells. Monocytes are cells that are able to quickly migrate
from the blood to site of infections where they differentiate into macrophages. They have
three functions: uptake of particles or pathogens and subsequent destruction, presentation
of pathogens to other cells of the immune system and secretion of soluble factors helping
resolve infections.
There are different techniques to measure these cell types:
The first method, the Coulter principle is a technology which is used to count and size
particles by using impedance measurements (Figure 1A). This principle is a very good
method to count blood cells in a short time. Nonconductive particles such as cells cause a
disruption in an electric field when they pass through the small opening between the
electrodes. These signals enable to distinguish different cell types.
In the second method, Fluorescence Activated Cell sorting (FACS) also called flow cytometry,
cells which were stained with fluorescently labelled antibodies, are passed through a
focused laser-beam where the labels are stimulated by the laser light (Figure 1B). The
fluorescent light (caused by antibodies coupled to antigens) and the scattered light (caused
by granules splitting the light) are detected separately.
In the third method a counting chamber (haemocytometer) is used to determine the number
of cells per unit volume in a suspension by counting the cells per square millimetre in a
lattice (Figure 1C). By dividing the total cells counted by the number of counted squares and
multiplying this number by 104 and the dilution factor the number of cells per millilitre (mL)
original suspension can be calculated.
The aim of this study was to analyse the variation of different methods to count white
blood cells in blood, spleen or lymph node (LN) cell suspensions of healthy mice.
Figure 1A: Principle of Coulter Counter
Nonconductive particles such as cells cause a
disruption in an electric field when they pass
through the small opening between the
electrodes. The changes in the electric field
are translated in signals that enable to
distinguish different cell types
# of cells counted
Figure 1B: Principle of FACS
Fluorescence activated cell sorting is based
on antigen-antibody reaction with
fluorescently labelled antibodies
x 104 x dilution factor (10) = # of cells/mL
# of squares
Figure 1C: Principle of Haemocytometer
The haemocytometer is used to count blood cells
under the microscope. Cells are dyed with Trypan
blue to distinguish live and dead cells. The amount of
cells per mL can be calculated with help of can be
calculated with help of the formula shown.
Methods
Preparation of blood for FACS analysis and haemocytometer: The blood was fixed with
1.25ml of 0.25% PFA and then the red blood cells were destroyed with lysing buffer. One
aliquot (5 µL), which was diluted in 1:10 Trypan blue, was taken to count in the
haemocytometer.
The Coulter Counter counts red blood cell parameters, therefore the blood was not lysed. An
aliquot of the untreated blood was directly analysed.
Preparation of spleen cells suspensions for all three counting methods: Spleens were
smashed with the plunger of a syringe and filtered through a 70 µm cell strainer and washed
with medium. The supernatant was removed and the red blood cells were destroyed with
lysing buffer. Cells were washed with a medium containing 10 % of fetal bovine serum (FBS)
and then resuspended in the same medium. An aliquot of cells was diluted 1:10 in Trypan
blue and counted in the haemocytometer. Another aliquot was analysed on a Beckman
Coulter counter.
Preparation of lymph node cell suspensions for all three counting methods: Lymph nodes
were smashed with the plunger of a syringe and filtered through a 70 µm cell strainer. Cells
were transferred to a 15 mL Falcon tube and washed with the medium. Then the cells were
resuspended in the medium and an aliquot of cells was diluted 1:10 in Trypan blue and
counted in the haemocytometer. Another aliquot was analysed on a Beckman Coulter
counter.
FACS staining: In a first step the Fc-receptors were blocked with an unlabelled anti-CD16/32
antibody then the cells were washed and a mix of antibodies (anti-CD19 FITC a marker for B
lymphocytes, anti-CD11b PE and anti-Gr-1 PerCP Cy5.5 in combination markers for
monocytes and anti-CD3 APC a marker for T lymphocytes). Cells were incubated for 20 min
on ice, washed twice and transferred into tubes containing Guava beads to determine
absolute cell number. The final concentration of the beads was 25’000 beads/mL. The tubes
containing the fluorescently labelled cells and beads were analysed on a BD FACS Calibur
flow cytometer.
Results
Table 1: Comparison of WBC, lymphocyte and monocyte counts in mouse blood, spleen or lymph node cell
suspensions analysed by three different methods.
Figure 2A: FACS analysis of mouse spleen cell suspension
FSC: forward scatter (approximates cell size); SSC: forward scatter (cell complexity or
granularity); CD19: B lymphocyte marker; CD3: T lymphocyte marker; Gr-1 and CD11b: double
positive marker for monocytes
Figure 2B: White blood cell counts
Number of white blood cells in blood, spleen
and lymph nodes counted with Coulter
Counter, FACS and haemocytometer
Figure 2C: Lymphocyte counts
Number of lymphocytes in blood spleen and
lymph nodes counted with Coulter Counter
and FACS
Figure 2D: Monocyte counts
Number of monocytes in blood, spleen and lymph
nodes counted with Coulter Counter and FACS
The number of cells in the blood counted by the Coulter Counter is very similar to the
amount of cells counted with FACS. The amounts of white blood cells in the lymph nodes
counted by the Coulter Counter, the FACS and with the haemocytometer are almost the
same. The difference between the numbers of cells in the spleen, counted with FACS and the
Coulter Counter, however is very big. For all three cell types FACS determines the highest cell
counts compared to the Coulter counter whereas counting spleen cell suspension in the
haemocytometer yields even higher number of total WBC.
Discussion
Counting cells on a haemocytometer is the cheapest method but has the disadvantage that
the different subsets of white blood cells (i.e. lymphocytes, monocytes, neutrophils,
eosinophils and basophils) cannot be distinguished. In addition this method is more labour
intensive if a lot of samples have to be analysed and the amount of cells has to be calculated
by hand.
The Coulter counter method is the quickest and still quite cheap method once the machine
has been acquired. But if the machine is inexistent in the lab, it is quite expensive because
the Coulter Counter has to be bought.
FACS is the most precise method if the cells have clear markers distinguishing them from the
other populations. In this study we had some conflicting results concerning the monocytes.
Using additional markers to clearly distinguish monocytes might be necessary. The
disadvantage of this method is that the fluorescently labelled antibodies and the machine to
read the cells are quite expensive. In addition this method is very labour intensive since the
cells need to be labelled before being analysed and it needs some experience to interpret
the results.
In lymph node cell suspensions all three methods can be used to count WBC and
lymphocytes. For monocytes further studies are required to find the most appropriate
antibody combination to distinguish them from granulocytes.
Conclusion
The Coulter seems to be reliable to grossly distinguish different WBC. It is quick and cheap
once the machine is available.
Gratitude
Thank you to Simone Zimmerli, my tutor during this week, and the entire personnel working
in the lab of Merck Serono for their help and support. I would also like to thank the
organisation “La science appelle les jeunes” for organizing this interesting week and giving
the opportunity to work in a professional lab.