EXPERIMENT 1: LYMPHOCYTE SEPARATION

TITLE: Lymphocyte Separation

OBJECTIVES: 1. To learn the techniques on how to separate the lymphocyte from full blood. 2. To observe the morphology of lymphocyte under microscope. 3. To observe the blood cells morphology without addition of staining dyes. 4. To determine the type of method used to conduct this experiment. 5. To identify the types of solutions and materials as well as its purposes in this experiment.

INTRODUCTION:

The blood consists of a fluid of complicated and variable composition, which is known as the plasma, in which contained the suspended erythrocytes, leucocytes and platelets. Leucocytes are mainly involved in immune system where it prevents infection. There are five types of leucocytes. They are neutrophils, eosinophils, basophils, monocytes, and lymphocytes. Lymphocyte is a type of white blood cell in the vertebrate immune system, which is a defense against the attack of pathogenic microorganisms such as viruses, bacteria, fungi and protista. Under the microscope, lymphocytes can be divided into large granular lymphocytes and small lymphocytes. Large granular lymphocytes include natural killer cells (NK cells) while small lymphocytes consist of T cells and B cells. A lymphocyte count is usually part of the peripheral complete blood-cell count and is expressed as percentage of lymphocytes to total white blood cells counted. A general increase in the number of lymphocytes is known as lymphocytosis whereas a decrease is lymphocytopenia.

Lymphocyte Separation Medium (LSM) is a sterile, iso-osmotic polysucrose and diatrizoate solution with low viscosity originally designed for the in vitro isolation of lymphocytes from diluted whole blood. LSM is based on the adapted method of isolating lymphocytes using centrifugation techniques by Boyum2 in which diluted blood is layered over the Ficoll-sodium metrizoate solution and centrifuged at a low speed for a short time. Centrifugation result in the formation of density specific layers. Lymphocytes are recovered by aspirating the plasma layer and then removing the cells. Excess platelets, LSM and plasma can then be removed by cell washing using phosphate buffer saline. Pure lymphocyte cell will be obtain and observed under microscope using 100x objective lenses. Differential migration following centrifugation will result in the formation of several cell layers. The heavier components cause the red blood cells to be forced to the bottom of the tube. The white blood cells and platelets are lighter, so they come to rest on top of heavier red blood cells in a layer called the buffy coat. Above the buffy coat rests the plasma. Mononuclear cells (lymphocytes and monocytes) and platelets are found at the plasma-LSM interface. Lymphocytes are recovered by aspirating the plasma layer and further washing removes the platelets, LSM and plasma.

MATERIALS: 15mL centrifuge tubes, microscope slides, cover slips, Pasteur pipettes, light microscope, test tube rack, micropipettes (100L, 1000L), blue tips, yellow tips, alcohol swabs, beaker, defibrinated human blood, lymphocyte separation medium (LSM), phosphate buffer saline (PBS) METHODS: 1. The blood from the EDTA tube was transferred into the 50 mL centrifuge tube. 2. The blood was diluted with equal volume of PBS. 3. The diluted blood was mixed gently by tilting the centrifuge tube upside down. 4. 5 mL of Ficoll paque solution was placed into 15 mL centrifuge tube. 5. The diluted blood on top of the Ficoll plaque solution was gently and slowly suspended until 12 mL by using a sterile Pasteur pipette 6. The solution was centrifuged at 2400 rpm for 40 minutes at 18C. 7. The upper layer was then removed. 8. The buffy coat was gently transferred from the solution into a clean centrifuge tube. 9. Then, the buffy coat was top up to 12 mL with PBS. 10. The mixture was then centrifuged at 4000 rpm at 18C for 15 minutes. 11. The supernatant was removed and the pellet can be stored at -80 C for future use.

RESULTS:

Plasma

Buffy coat

Red Blood Cell

Diagram 1: Separation of whole blood after centrifugation into three layers: plasma, buffy coat and cellular components.

Red Blood Cell

Diagram 2: Before the separation of defibrinated human blood (with only 3mL of PBS)

Red Blood Cell

Diagram 3: Before the separation of defibrinated human blood (with more PBS to dilute it)

Lymphocyte

Red Blood Cell

Diagram 4: Observation of lymphocyte after centrifugation with phosphate buffer saline or after the separation of defibrinated human blood under oil immersion magnification (100x).

DISCUSSION: Based on Diagram 2 (before the separation of defibrinated human blood with only 3mL PBS), lymphocytes are hardly to be seen and red blood cells are mostly seen. This situation is known as the low yield of lymphocytes with normal viability. This may be due to the blood is not diluted 1:1 with the balanced salt solution, phosphate buffer saline (PBS). It is unusually high hematocrit. Hematocrit is the proportion of blood volume that is occupied by red blood cells. The high cell density results in large numbers of lymphocytes being trapped by red blood cell aggregates. In order to overcome this problem, the human blood sample can be diluted further with the PBS. Dilution gives a better lymphocyte yield and reduces the size of the red blood cell clumps. Based on Diagram 3 (before the separation of defibrinated human blood with more PBS), lymphocyte yield is clearer and few lymphocytes can be seen. On the other hand, based on Diagram 4, the lymphocytes can be seen but not as clear as expected. This is because the human blood sample had undergone the centrifugation in the optimum temperature that is from 180C to 200C. Aggregation of erythrocytes is enhanced at higher temperatures (370C) which consequently decreases the yield of lymphocytes. At lower temperature (40C), the rate of aggregation is decreased but the time of separation is increased which also decreases the yield of lymphocytes. In Diagram 4, few of the lymphocytes can be clearly seen. The lymphocytes are stained in slightly in purple colour while the cells that surround the lymphocytes are red blood cells, which are red in colour. In order to reach the maximum separation of lymphocytes from the human blood, there is several ways that can be used. Firstly, the blood samples should be processed as soon as possible after collection to ensure optimal results. Storage of blood samples for 24 hours at room temperature results in reduced lymphocyte yield. Secondly, the human blood sample can be diluted with balanced salt solution in order to have a better lymphocyte yield and reduces the size of red blood cell clumps. This is because some lymphocytes are trapped in the clumps and sediment with the erythrocytes when the erythrocytes in whole blood are aggregated. Besides that, temperature also plays important role in the maximum separation of lymphocytes from

the human blood. Too high or too low temperature will affect the lymphocyte yield. Thus, optimum temperature for the separation of lymphocytes from the human blood is 180C- 200C. In this experiment, there is several safety precautions need to be taken. The tip of the micropipette must be changed after it was used to transfer the sample. This is to avoid contamination (mixing with another sample). The plunger of the micropipette must be depressed to the first stop before dipping the tip into the solution so that there will be no bubbles present. Besides that, we must wear gloves when we are handling the human blood sample because the human blood sample may be contagious or infectious. Other than that, the Pasteur pipette must be changed after transfer each sample in order to prevent the contamination. In order to create a sharp blood-LSM interface, the centrifuge tube which is containing LSM must be slanted about 450 and carefully layers the diluted human blood sample over the LSM. In addition, phosphate buffer saline (PBS) is used to dilute the blood sample instead of the distilled water. This is because PBS contains sodium chloride, sodium phosphate, potassium chloride and potassium phosphate which help to maintain a constant pH of the cell. Lat but not least, PBS with EDTA is used to disengage attached and clumped red blood cells.

CONCLUSION: From this experiment, the separation technique of the lymphocyte using Ficoll-sodium diatrizoate is learned and the internal structures of the lymphocyte using the light microscope are studied. By using Ficoll-sodium diatrizoate, erythrocytes are sediment to the bottom of the tube while lymphocytes are collected from the interface between the two phases. Lymphocyte has a large, dark-staining nucleus with little or no cytoplasm. Mostly the lymphocytes will stain with purple colour. However, this technique may sometimes results is inaccurate due to human errors. Therefore, the precautions should be taken into consideration in order to prevent the results of the experiment from being affected.

REFERENCES: 1. Giorgio Carboni (1997). The Blood Cells. Retrieved on 18 June, 2011 from Fun Science Gallery web site: http://www.funsci.com/fun3_en/blood/blood.htm 2. Krackeler Scientific, Inc. (2009). Cell Separation: Lymphocyte Separation Medium (LSM). Retrieved on 19 June 2011 from Google web site:
http://www.krackeler.com/products/1454-Serum-Free-Specialty-Media/14481-Cell-

Separation-Lymphocyte-Separation-Medium-LSM-.htm 3. Boyum, A Isolation of mononuclear cells and granulocytes from human blood. Scand.J.Clin.Lab Invest. 21,Suppl.97:77,1968