This document provides instructions for examining blood samples. It describes the components of blood and how to collect blood through either capillary or venous methods. Detailed procedures are provided for making wet blood preparations, thick blood films, and thin blood films in order to examine blood cells and detect parasites. Quality controls and common problems in blood film preparation are also discussed.

1. Examination of blood
Prepared by: Mathew T. Moses
RHSI 2015

2. General concept of blood
• Blood consists of two parts:
• Cellular part which consists of three types of
cells: RBCs, WBCs and platelets. (45%)
• Fluid part which consists of plasma and serum.
(55%)
• many diseases of the body are recognized by:
1- changes in the appearance and numbers of blood
cells;
2- changes in the composition of plasma; and
3- identification of infectious organisms in cells of
plasma.

3. Cont. …
• The average adult has a blood volume of
roughly 5 liters.
Cells: One microliter of blood contains:
• 4.7 to 6.1 million (male),
4.2 to 5.4 million (female) erythrocytes/RBCs.
• 4,000–11,000 leucocytes/WBCs
• 200,000–500,000 thrombocytes/platelets

4. Plasma
• The blood plasma volume totals of 2.7–3.0 liters in an
average human.
Important components of plasma include:
• Serum albumin
• Blood-clotting factors (to facilitate coagulation)
• Immunoglobulins (antibodies)
• lipoprotein particles
• Various other proteins
• Various electrolytes (mainly sodium and chloride)
Serum: refers to plasma from which the clotting proteins have
been removed. Most of the proteins remaining are albumin
and immunoglobulins.

5. Functions of blood
• Supply of oxygen to tissues
• Supply of nutrients such as glucose, amino acids, and fatty
acids
• Removal of waste such as carbon dioxide, urea, and lactic acid
• Immunological functions, including circulation of white blood
cells, and detection of foreign material by antibodies
• Coagulation, the response to a broken blood vessel, the
conversion of blood from a liquid to a semi-solid gel to stop
bleeding.
• Messenger functions, including the transport of hormones
and the signaling of tissue damage
• Regulation of body pH
• Regulation of core body temperature
• Hydraulic functions

6. Blood collection
Capillary blood:
• Principle: collect capillary blood from the side
of the finger in adults and children.
• Avoid the tip of the finger as it is very sensitive
• In Infants, collect capillary blood from the side
of the heel.
• Transfer the capillary blood directly onto clean
glass slides or into 20ul glass pipettes.

7. Requirements
• Sterile lancet
• Dry cotton wool
• Alcohol swab
• Clean glass slides of 20ul pipettes.

8. Procedures
1. Clean the skin thoroughly at the selected site
using alcohol swab, allow the skin to dry.
2. Prick the skin firmly so that blood flows freely
without excessive squeezing.
3. Wipe off the first drop with a plug of dry cotton
wool. The first drop is contaminated with dirt
and tissue fluid.
4. Squeeze the skin gently and collect the next
drop on to a slide or into a pipette. With small
children it may be easier to fill the pipette from
a large drop on a slide.

9. Cont. …
• Apply pressure to the finger prick with a plug
of dry cotton wool.
• Immediately place the lancet in safety box.
• Place the cotton wool in the bucket marked
‘INCENIRATION’

10. Venous blood
• Principle: collect venous blood from a vein in
the crease of the elbow, with the arm out-
stretched.
• In adults, if the is not possible; use a vein in
the forearm or back of the hand.
• In babies and small infants, collect blood from
a vein in the back of the hand or ask the
clinician to take a sample from scalp vein.

11. Requirements
• Dry cotton wool
• Alcohol swab
• Tourniquet or blood pressure cuff
• Sterilized dry syringe and needle (G 21 or G
23)
• EDTA or plain blood container as required

12. Procedures
1. Allow the patient to sit comfortably with one arm
resting on the bench or table. Fit the needle on to the
syringe.
2. Apply the tourniquet to the upper arm. Ask the
patient to open and close the fist several times.
3. Disinfect the skin over the vein using alcohol swab,
allow the skin to dry.
4. With the bevel of the needle facing up, carefully
insert the needle into the vein. Slowly draw out the
plunger until the required amount of blood fills the
syringe. Release the tourniquet or blood pressure cuff,
then withdraw the the needle from the vein and
immediately apply pressure on the puncture site with
plug of dry cotton wool.

13. Cont. …
5. Detach the needle from the syringe and
transfer the blood sample into the correct
specimen bottle. Label each bottle with the
patient’s laboratory number using a grease
pencil.
6. Immediately replace the needle on to the
syringe and place it in safety box.
7. Place used cotton wool in the bucket marked
‘INCENIRATION’.

14. Preparation of blood films
• Use capillary or anti-coagulated venous blood.
• Do not use clotted blood for making blood
films because blood cells and parasites are
trapped in the clot.
• Do not use blood anti-coagulated with
heparin for preparing thick and thin blood
films because heparin cause:
• Blue-background staining, clumping of WBCs
and platelets, and poor staining of white cells.

15. Wet preparation
Principle: a wet preparation of blood is a drop of
fresh blood placed on a glass slide and covered
with a coverslip. The coverslip spreads the blood
cells evenly.
Value:
• Detection of large motile parasite in blood
such as: microfilaria of Wuchereria bancrofti,
Loa loa and Mansonella perstans
• Sickle cell screening.

16. Procedures:
• Clean a glass slide with gauze or dry cotton wool.
• Collect a sample of capillary or anti-coagulated
venous blood.
• When using capillary blood, carefully touch the
drop of blood on to the center of the slide,
making sure the slide does not touch the kin.
• When using anti-coagulated venous blood, mix
well and place a small drop of blood on to the
center of the slide using a clean Pasteur pipette
and rubber teat. Label the slide with the patient’s
lab No. using a grease pencil.

17. Cont. …
• For detection of motile parasites, put on
coverslip and examine without delay after you
mix the drop of blood with normal saline.
• Report your finding.

18. Thick blood film
• Is a small drop of fresh blood which is spread
in a circle on a glass slide and allow to dry. It
consists of many layers of blood cells on top of
each other. Thick blood film is used to detect:
• Plasmodium species (malaria parasites)
• Borrelia species
• Trypanosomes
• Microfilariae

19. Procedure
• Clean a glass slide with gauze or dry cotton
wool.
• Collect a sample of capillary by touching the
drop of blood on to the center of the slide
making sure the slide does not touch the skin,
or anti-coagulated venous blood by mixing
thoroughly and place a small drop of blood on
to the center of the slide using a clean Pasteur
pipette and rubber teat.

20. Cont. …
• Spread the blood in a circle approximately 1 cm in
diameter using the corner of another slide. The
film is the correct thickness when newsprint or
the hands of a watch can be seen through it.
Label the slide using a grease pencil.
• Air dry the film in a horizontal position. Do not
dry the film by heating over a flame or placing on
a hot subject.
• Label the slide with the patient’s lab number.
• Stain using the Feld stain technique or Giemsa
stain technique.

21. Thin blood film
• Is a small drop of blood that is spread thinly
on a glass slide so that red cells do not
overlap.
A thin film is used for:
• Differential white cell count.
• Examination of blood cells morphology
• Clear identification of blood parasites.

22. Procedures
• Clean a glass slide with gauze soaked in soluble
methanol. Allow to dry.
• Collect a sample of capillary or anti-coagulated
venous blood.
• When using capillary blood, carefully touch the
drop of blood on to the slide from one end,
making sure that the slide does not touch the
patient’s skin. When using anti-coagulated
venous blood, mix thoroughly and place a small
drop of blood on to the slide from one end, using
a clean Pasteur pipette and rubber teat. Place the
slide horizontally on a flat surface.

23. Cont. …
• Hold a spreader (or coverslip) in the center of
the slide at an angle of 500.
• Move the spreader back until it is in contact
with the blood, and allow the blood to spread
along the base of the spreader.
• Now reduce the angle of the spreader to
about 300 and move the spreader steadily
across the slide. A good thin film should end in
a conical ‘tail’ and should cover 2/3 of the slide

24. Cont. …
• If the drop of the blood is too large, transfer a
small portion of blood by touching it with the
edge of the spreader and start the film in a fresh
place ahead of the large drop.
• If the blood is anemic, hold the spreader at a
greater angle and move it faster. This makes a
slightly thicker film.
• Dry the film quickly by waving it in the air. Slow
drying results in shrinkage of red cells. Do not dry
the film by heating over a flame or placing on a
hot object.

25. Cont. …
• Label the slide with patient’s lab number by
writing with lead pencil across the thickest
part of the film or on the frosted end of the
slide.
• Fix the film by dipping for 2 seconds in
absolute methanol. Allow to dry.
• Stain using the reverse field stain technique or
Giemsa stain

26. Quality controls on blood films
• A thick and thin blood film may be made on
the same slide.
• It useful when examining blood film for
malaria parasites.
• The thick film is examined to establish the
presence of parasites, and the thin film is used
for species identification
• Make the thick film at one end of the slide.

27. Cont. …
• Spread the thin film starting from the center
and moving towards the other end of the
slide.
• Allow the film to dry
• Stain the thick film using the field stain
technique. Allow to dry.
• Fix the thin film in absolute methanol.
• Stain the film separately, using the reverse
field stain technique or Giemsa stain.

28. Common problems encountered
when making thin film are:
• The film is too thick and the end of the film is
lost. This occurs when the drop of blood is too
large.
• The blood clots while the film is being
prepared. This occurs when capillary blood is
left too long on the slide before spreading.
• The film has an irregular tail. This occurs when
a ragged spreader is used or there is uneven
contact between spreader and slide

29. Cont. …
• The film is too thin. This occurs when the drop
of the blood is too small and the film is spread
to slowly;
• The film does not adhere o the slide. This
occurs if the slide is greasy and and has not
been adequately cleaned with alcohol