Blood Tests

1. Blood Tests Learning objectives: (a) describe how blood samples are taken and blood smears (films) are made; (b) describe the procedure for the differential staining of blood smears to show leucocytes;

2. Why take blood? • Provides valuable information about health • Can help to diagnose conditions • If a large enough sample of blood is taken at once, several different tests can be carried out.

3. How to take blood • Blood is commonly taken from a vein, at the elbow. • Veins take blood back to the heart at a much lower velocity and pressure than arteries. • A tourniquet is applied to the upper arm to make the vein stand out – this allows the needle to go into the vein easily. • The area round about the arm is cleaned with a disinfectant (alcohol based) solution. • The needle, with a sterile syringe attached, is pushed into the arm. • The plunger is pulled back, sucking blood out of the vein. • When enough blood is collected, the needle is removed and a small ball of cotton wool is applied with pressure to the area to stem any blood flow. • A dressing is applied to the wound

4. Making and staining a blood film Small drop of blood is placed near the end of the microscope slide The end of another slide is placed on the sample slide The spreader is held at an angle of about 30o and pushed along the slide, spreading the drop of blood as a smear The slide is labelled and allowed to dry in the air, so the cells stick to the slide Slide is stained using Romanowsky stain (Leishman’s stain). This is poured on and left for 2 minutes before being washed off with water The slide is fixed with alcohol to preserve the cells

5. Your task – start in class, to be completed as homework Make an information poster on how to make and stain a blood film OR How to take a blood sample. Your poster should be colourful, clear and have full instructions that include diagrams. Best poster gets a prize and the chance to gloat to all the losers 

6. Using a haemocytometer Learning objectives: (c)describe the use of a haemocytometer to count the numbers of erythrocytes and leucocytes (to include details of dilution);

7. A haemocytometer • A haemocytometer is the name given to a counting chamber designed to count the number of blood cells in a blood sample. • A haemocytometer has a central platform with grooves either side. • There is also a grid attached, which resembles graph paper. • The total volume of a haemocytometer is exactly 0.004mm3

8. Corner squares are used to count leucocytes (WBCs) The triple lined squares are used to count erythrocytes (RBCs)

9. Counting erythrocytes (RBCs) • Prepare a blood dilution, using a blood pipette - graduated to 0.5, 1 and 101. - Take up blood to the 0.5 mark. 2. Mix the blood with the dilution fluid (known as Dacie's fluid). Take up to the 101 mark. 3. There is a bulge in the middle of the of the pipette which creates a dilution of 1 in 200. 4. Run the first few drops out of the pipette, these will be discarded. Then place 1mm3 of the diluted blood on the haemocytometer.

10. Counting erythrocytes North 5. You need to randomly count all erythrocytes that appear in 5 of the triple lined squares. 6. You do not count all cells on the borders of the triple lined squares unless they follow the northwestrule. West

11. Counting erythrocytes 7. Remembering each triple lined square has a volume of 0.004mm3 the fivecombined is 0.02mm3 8. Now we can calculate the erythrocyte count of the individual in 1mm3 of blood

12. Counting erythrocytes • The volume of one triple lined square is 0.004mm3 • The blood was diluted 200 times • The total volume of 5 triple lined squares was 0.02mm3 • Assume E is the number of erythrocytes counted. • To calculate the number of RBCs in 1mm3 1/0.02 x E x 200 OR E x 10,000 If the total erythrocyte count from the haemocytometer was 64 what would the total erythrocyte count be? 64 x 10000 = 640,000

13. Calculations • Calculate the following: • 54 erythrocytes counted over 5 triple lined squares • 77 erythrocytes counted over 5 triple lined squares • 49 erythrocytes counted over 5 triple lined squares • 34 erythrocytes counted over 3 triple lined squares • 40 eryhtrocytes counted over 4 triple lined squares

14. Homework • Produce a similar procedure for counting leucocytes. • Use diagramsto explain the procedure

15. Counting Leucocytes • Done using a white blood cell pipette • Similar to red blood cell pipette but is graduated 0.5, 1 and 11 mark and mixed together using diluting fluid. • This means the blood is diluted by a factor of 20 • This causes the RBCs to burst but the WBCs to remain intact.

16. Corner squares are used to count leucocytes (WBCs) The triple lined squares are used to count erythrocytes (RBCs)

17. Counting Leucocytes • The first 1/3 of the mixture is allowed to run out – this is discarded. • The mixture is added to the haemocytometer, but the larger squares on the grid are used this time. • Larger squares measure 1 x 1mm and the depth of the haemocytometer is 0.1,, • Therefore 1x1x0.1 = 0.1 mm3 • WBCs in the four corners are counted. The total volume of the 4 squares is 0.4mm3

18. Counting Leucocytes • If the number of cells counted in all four squares = L, then the number of leucocytes per mm3of the original blood sample: • = 1/0.4 x L x 20 • = 50 x L