Blood Cells In Inflammation

Question 1

What materials are needed to analyse a blood sample for cells in inflammation?

Answer 1

ECTA blood sample, crystal violet solution, haemocytometer, Gilson pipette, eppendorf tubes, microscope

Question 2

What is the method for detecting blood cells in inflammation?

Answer 2

1. add 180 µL of crystal violet to eppendorf tube
2. take 20µL of EDTA blood, mix with crystal violet, wait 2 mins
3. load haemocytometer chamber
4. count number of WBC’s

Question 3

How to count the number of blood cells in a haemocytometer chamber?

Answer 3

Allocate a square to be counted, (normally corners and middle)

1. count the number of blood cells i.e. 31, 3 non-viable
2. count the number of viable cells i.e. 28/31x100 = 90.3%
3. take an average number of cells per square = viable cells/squares i.e 28/1 = 28
4. calculate the dilution factor = final volume/volume of cells 200/100 = 2
5. Calculate the concentration = avg# cells/square x dilution factor x10^4 = 28x2x10^4= 560000 or 5.6 x10^5 viable cells/mL

Question 4

How to isolate leukocytes?

Answer 4

Take required volume of blood from jugular vein
Add immediately to tube containing anti-coagulant
- Heamocrit is the % of whole blood that is red blood cells
- pipette off white blood cell layer
- density gradient is needed (thicoll, percoll or histopaque) when centrifuged

Question 5

How to isolate WBC’s?

Answer 5

Diluting blood with PBS
Layer diluted blood on top of percoll/histopaque
Centrifuge