Isolation of leukocytes

Question 1

How can separation methods of leukocytes from blood be divided?

Answer 1

Non-specific
Function
Specific

Question 2

What are the non-specific isolation methods based on?

Answer 2

Difference in physical properties (size, density and electric charge)

Question 3

What are the function separation methods based on?

Answer 3

Adherence abilities

Question 4

How are leukocytes separated according to density?

Answer 4

Gradients are prepared using sucrose, Ficoll or Percoll

Question 5

Which cells are separated according to adherence abilities?

Answer 5

Monocytes, macrophages, B-cells and T-cells

Question 6

What is specific separation methods of leukocytes based on?

Answer 6

Presence of specific markers (CD markers) incorporated into cell membranes

Question 7

How is specific separation of leukocytes carried out?

Answer 7

Monoclonal antibodies are labelled with specific beads, which will bind to CD markers present only on the membranes of target cells

Question 8

What is positive separation in specific separation of leukocytes?

Answer 8

Separate required cell subpopulations

Question 9

What is negative separation in specific separation of leukocytes?

Answer 9

Removal of unwanted cell subpopulations

Question 10

How are leukocytes isolated from whole blood using osmotic lysis?

Answer 10

Short exposure of blood to distilled water at ratio 1:2 for 35-40 seconds. ‘
Immediately add 2.7% NaCl solution to make solution isotonic.
Centrifuge cell suspension.
Remove supernatant and wash pellet of cells with PBS and repeat twice.
Resuspend in Hank’s balanced salt solution.

Question 11

What is left after removing of red blood cells from blood by osmotic lysis?

Answer 11

all leukocytes

Question 12

What happens when you use cell separating solution on blood?

Answer 12

Mononuclear leukocytes and polymorphonuclear leukocytes in sediment with red blood cells

Question 13

How can monocytes be separated from mononuclear leukocytes?

Answer 13

They adhere very well to plastic or glass surface

Question 14

How can T and B lymphocytes be separated from mononuclear leukocytes?

Answer 14

Using anti-CD3 and anti-CD19 mAb labelled with magnetic beads

Question 15

How can lymphocytes be separated from mononuclear leukocytes?

Answer 15

They remain after removing of monocytes

Question 16

How is leukocytes separated from whole blood using lysing solution?

Answer 16

Blood is exposed to lysing solution for 10 to 15 minutes.
Red blood cells are destroyed, but leukocytes are not affected.
Cell suspension is then centrifuged.
Supernatant is removed and pellet is washed with PBS, repeat twice.
Cells are resuspended in Hank’s balanced salt solution.

Question 17

What is the procedure of isolating leukocytes from whole blood using lysing solution?

Answer 17

Removal of red blood cells after labelling of leukocytes for analysis in a flow cytometer

Question 18

How are mononuclear leukocytes isolated from whole blood using cell-separating solution?

Answer 18

Diluted blood is added on top of separating solution.
Blood cells are separated during gradient centrifugation according to their density into layers.
Mononuclear leukocytes and platelets will remain on the top of the separating solution as they are less dense.
Red blood cells and polymorphonuclear cells will be under the separating solution as they are more dense.

Question 19

What is the density of the separating solution used to separate mononuclear leukocytes from whole blood?

Answer 19

1.077 g/cm^3

Question 20

How many mononuclear leukocytes can be separated from whole blood?

Answer 20

1-2 x 10^6 /ml

Question 21

What should the viability of separated mononuclear leukocytes be?

Answer 21

94-100%

Question 22

What are the methods of isolation of leukocytes by magnetic separation?

Answer 22

Tube-based method and column-based method

Question 23

What is the principle of separation of leukocytes by magnetic separation?

Answer 23

Mixing cells with paramagnetic beads that only exhibit properties when placed within a magnetic field, which are coated with an antibody

Question 24

What happens in the tube-based method of isolation of leukocytes by magnetic separation?

Answer 24

Target-bead complexes are removed from the cell suspension using external magnet that draws complexes to the edges of the test tube, allowing the supernatant to be removed. Removing the magnetic field allows resuspension of the target-bead complexes. Separation is gentle and do not require centrifugation or columns.