Lund University Immunotechnology Dept.

Characterization of leucocytes in
PERIPHERAL BLOOD CELLS

Student: Mahesh Bhupathi Pardhasarthy Supervisor: Henrik Johansson Sandra Sernbo

INTRODUCTION

During this experiment we have learnt about different types of white blood cells through various techniques; they are counting cells through burker chamber, immuno cytochemistry, Flow cytometry, staining of blood cells from the blood sample collected from an individual
COUNTING CELLS IN A BURKER CHAMBER

This technique allow us to count the number of viable cells in the blood based on the differential staining of the dead cells and the viable cells based on their membrane integrity , when the cells are stained with trypan blue the dead cells will appear in blue color because of damaged membrane integrity while the viable cells remain unstained , based on the staining we count the number of cells using a specialized counting chamber called burker chamber , each chamber is divided into counting fields which contains 9 small squares say A-square and which in turn are divided in to 16 small squares say B- square and each A square can accommodate 0,1 micro liters of sample The amount of viable cells =viable cells in single A-square * 104* dilution factor
FLOW CYTOMETRY

Flow cytometry is a way of analyzing the physical and a few chemical characters of cell through using optics, a typical flow cytometry consists of a monochromatic beam source which is usually a laser beam and light analyzer which analyze the laser beam, each cell is allowed to pass through a sensing point where laser beam is focused on to the cell and the laser beam past the cell which is scattered to 2 lenses fixed at right angles to the sensing point one for sensing the fluorescence and the other for measuring the morphological characters of the cell which is eventually measured by analyzing the forward scattered beam and the side scattered beam which is a measure of , cells size and surface morphoogy respectively , the instrument can also be used for cell sorting by passing the cells across a charged surface, when the cells are stained with certain stains that are linked to an antibody which is specific for a specific antigen of the cell , then the cellular composition can be reviewed in a multitude of cells, usually we use fluorescent dyes such as fluorescent (FITC) and phycoerythrin (PE) for staining the cells and depending on the requirements more complex fluorescent dyes may be used, the data obtained from the cytometry have details about cell size,(forward scattering), cell complexity (side scattering), cellular characters and function
MAY-GRUNW ALD-GIESMA STAINING

This is a staining technique based on the type of the cytoplasmic granules in the cell usually the eosinphils contains acidic granules that are stained by acidic dyes while the basic dyes used for staining basophils as they have basic granules while the neutrophils are stained by neutral dyes and the nucleus is stained by DAPI as that stains all the negatively charged particles like DNA hence nucleus can be visualized by this techniques , the staining indeed occurs due to the electrostatic

interactions of the dyes to the ligands hence we can visualize all the granulocytes and also the nucleated cells with this technique, in general blood consists of 99% of erythrocyte population and the rest is leucocytes and thrombocytes
IMMUNOCYTO CHEMISTRY

Immuno cytochemistry is widely used to study, or diagnosis of different cells, we use different antibodies targeting to the specific antigens of the cells, in order to visualize them they are coupled to florophore which can be seen under a fluorescence microscopy. In this experiment we have used HLADR and CD19 recognizing antibodies to distinctly distinguish between APC form the other cells and in between them
MATERIALS AND METHODS

We have done everything according to manual except some corrections like, dividing the cells to 600ml to fraction B and 200ml to fraction A in step -4 flowcytometry, and we used CD14 PE instead of FITC in tube 2 and CD14 PE instead of CD3 APC and some changes in the fractions of dilutions in the May-gurnwald-giemsa method and Immuno Cytochemistry
RESULTS FLOW CYTOMETRY

The report from the flow cytometry is represented in the form of graphs which were appended at the end and all the combined data based on the markers in conjunction to their marked cells has been represented in the following table.

Marker B-cell
CD-14 CD-19
HLA-DR

Naive Bcell + + +

T-cell

Type of cell Naive T- TH-cell cell

TC-cell

Monocytes +

Ig-D CD-3 CD-4

CD-45RA % of gated

+ + +

+ + + + 56%T + + 8 +

62%B

17

33

Table:1 showing the distribution of leucocytes in the PBMC and percentage with respect to their markers

IMMUNO CYTOCHEMISTRY

All the samples when observed under fluorescence microscope they show blue nucleus while the samples from control shown only nucleus while the other sample has shown some green cells indicating Monocytes and some cells with both green and red light indicating B cells

Fig-1: Image from fluorescent microscopy showing all the B-cells, Monocytes and Control MAY-GRUNWALD-GIEMSA METHOD

Fig-2: Image showing various cells stained differently with May-grunwald-giemsa method

DISCUSSION

FLOW CYTOMETRY

The results from the flowcytometry with the tube A has showed 3 distinct population with forward scattering indicating the cells on the top will be the lager cells and a medium sized cells in the middle and small cells or particles and perhaps they could be the cell debris The cells with the tube 2 has show three gates they are gate-3 and gate-4 and gate-5(refer to appendix for the charts) and gate-3 shown a positive population for Ig-D and CD-14 PE-A and which indicates Monocytes and gate-5 has shown positive for CD-19 hence it is B-cell population while the cells in the gate 4 are negative for both CD19 and CD14 hence they can be T-cells and NK cells ,while gate 7 is positive for HLADR preCP-Cy5-5-A and negative for CD-19 indicating Monocytes population while gate 6 is positive for CD19 APC-A which is B-cell population cells, gate 8 is double negative for HLA-DR and CD19 whichi is again T cells and NK-cells, cells in gate 10 are positive for CD19 but negative for IgD CD14 PE A hence it is Memory Bcell and the cells of gate 9 are positive for IgD or CD19 hence it is nave B-Cell The cells from tube 3 shown gates 11 that is entire T-cell population as it has both CD3 positive cells and CD 4 positive cells and cells with CD3 positive and CD4 negative cells indicating TC cells hence gate 12 is the population of cells THand gate 13 Is the population of TC cells. Tube B it has shown gate 14 which is positive for CD14 and negative for CD3 indicating it to be a monocyte and gate 15 is negative for both CD 3 and CD 14 indicating it as complete absence of Tcells as the percentage of population bearing this is zero and hence we can say that the T cells are completely absent in them The markers CD14 is a coreceptor for bacterial lipo poly saccharide, and that was only found in Monocytes, CD 19 is a coreceptor in B cell that works in conjunction with CD 21 and CD 81 , the HLA DR which is a surface receptor of MHC II molecule, IgD is found on nave b cells and also on Plasma B cells, CD3 and CD4 are found on the surface of tcells but CD4 is only found in TH cells, CD45RA is found in nave T-cells
IMMUNO CYTOCHEMISTRY

Since all the nuclei has been stained by DAPI , hence we can clearly see all the nucleated cells in blue colur but B-cells have CD20 and HLA-DR on their surface and hence when they are treated with antibodies specific for CD20 and HLADR and which are eventually attacked by goat mouseAlexa568 and SA488 , hence they glow both green and red but the Monocytes can only express HLA-DR on to their surface hence they show only green colour, but during the laboration we has

done 3 steps for blocking affecting the sensitivty of the method they are blocking with fetal calf serum and goat serum which contain a great deal of protiens and antibodies that compete for any surface Ig-receptors and also prevents any unspecific binding, the second blocking step is done with avidin and biotin to prevent the unspecific intreaction with streptavidin and also to block endogenous biotin and the third step of blocking is to prevent the free variable arm of the mouse antibody that is added in the first step binding to the mouse antibody HLA-DR , hence that blocking is done.
MAY-GRUNWALD-GIEMSA STAINING METHOD

Staining the blood cells with May-grunwald-giemsa staining method has let us to visualize granulocytes and agranulocytes and also the poly morphism of various nucleuii from different leucocytes the results has clearly shown these cells,
NEUTROPHILS: with clear cytoplasm, and multi lobed nucleus, since we have not used any neutral

dyes hence we can see a clear cytoplasm.

EOSINOPHILS: though clear distinction cannot be made by analysing the red cytoplasmic granules

but one can judge the presence of eosinophils by seeing at the bilobed nucleus.
BASOPHILS: from figure 2 in results we can see the basophils can be seen with blue coloured

granules in the entire cytoplasm

LYMPHOCYTE: form the figure it can be seen the lymphocytes with a clear circular large nuclei

with little cytoplasm.

MONOCYTES: these cells can be analysed with a kidney shaped nucleus.

REFERENCE

 

Henrik johansson, Sandra Sernbo. Characterization of luecocytes present in pheripheral blood , Lund university Janeways IMMUNO BIOLOGY

APPENDIX