Preparation of blood film Dalia Kamal Eldien MSc in microbiology Practical NO-4-

Introduction  Types of blood specimen include  Venous blood  Capillary blood

Blood collection techniques Capillary blood obtained by finger stick: 1. Label pre-cleaned slides (preferably frosted-end) with patient’s name (or other identifier), date and time of collection. 2. Wear gloves. 3. Clean slides with 70 to 90% alcohol and allow to dry. Do not touch the surface of the slide where the blood smear will be made. 4. Select the finger to puncture, usually the middle or ring finger. In infants, puncture the heel.

Blood collection techniques 5. Clean the area to be punctured with 70% alcohol; allow to dry. 6. Puncture the side of the finger ball, or in infants puncture the heel. 7. Wipe away the first drop of blood with clean gauze. 8. Touch the next drop of blood with a clean slide. Repeat with several slides (at least two thick and two thin smears should be made).

Capillary blood from adult

Capillary blood from infant

Venous blood  Identify the patient  Applying the tourniquet  Select a suitable site for venipuncture.  Disinfect the site by 70% alcohol  Attach needle to holder  Place tube into holder foe the evacuated tube  Insert needle and Perform the venipuncture.  Collect the sample in the appropriate container.  Apply Pressure and adhesive bandage

Anatomy of vein

Evacuated blood tube

Blood container

 For perfect malaria staining, thin and thick films should be made on separate slides  Thin films will be fixed with Methanol 15 – 30 seconds, are made by placing a drop of blood on one end of a slide, and using a spreader slide or by automated slid- spreader to disperse the blood over the slide's length. Blood film preparation

 Thick films:- place a drop of blood in the middle of a clean microscope slide and with the corner of a second slide spread the drop until it is about the size of a five cent coin. should not be fixed

Automated slid-spreader

 A good thin smear should….. Cover around ¾ of the slide Have a feathered edge Move from thick to thin Cells should be evenly distributed RBCs should not overlap  Factors that affect the smear include….. Blood drop size Angle of the slide used to spread Speed of the procedure

Steps to make a thin blood film

Blood film

Thick blood film

Thick& thin blood film

Staining blood film  Routine analysis of blood in medical laboratories is usually performed on blood films stained with Romanowsky, Field’s, Wright's, or Giemsa stain. Wright-Giemsa combination stain is also a popular choice.

Staining thin blood films with Giemsa  Prepare 6% Giemsa solution staining solution:  Take 94 ml buffer solution pH 7.2  Then add 6 ml Giemsa stock solution  Fill staining dish with staining solution  Place thin film and thick films into the staining dish.  Stain blood slides for 45 minutes  Wash the slides with tap water. Thick films need careful rinsing! (since they are not fixed before staining)  Let air dry or use heating plate

Giemsa stain

Field’s stain method for thick blood films  The Field’s stain is made from 2 components. Field’s A is a buffered solution of azure dye and Field’s B is a buffered solution of eosin. Both Field’s A and B are supplied ready for use by the manufacturer. Method 1. Place a drop of blood on a microscope slide and spread to make an area of approximately 1 cm2. It should just be possible to read small print through a thick film. 2. The film is air dried and NOT fixed in methanol.

3. The slide is dipped into Field’s stain A for 3 seconds. 4. The slide is then dipped into tap water for 3 seconds and gently agitated. 5. The slide is dipped into Field’s stain B for 3 seconds and washed gently in tap water for a few seconds until the excess stain is removed. 6. The slide is drained vertically and left to dry. 7. Examine under the microscope by X100

Field’s stain