EXAMINATION OF BLOODDone by: Miss Nada

EXAMINATION OF BLOOD

Examining the specimen of blood

microscopically for:

1. Plasmodium spp. ( Malaria parasites).

2. Trypanosoma spp.

3. Microfilaria spp.

WHAT IS MALARIA? Malaria is a vector-borne infectious disease caused by single-celled protozoan parasites of the genus Plasmodium.

Malaria is transmitted from person to person by the bite of an infected female Anopheles mosquitoes.

WHAT IS MALARIA? Infection can also occur by transfusion of infected donor blood, by injection through the use of needles contaminated with infected blood.

PLASMODIUM SPECIES

1. Plasmodia falciparum,

2. Plasmodia vivax,

3. Plasmodia malariae and

4. Plasmodia ovale.

The most dangerous of the four is

P.falciparum.

LABORATORY DIAGNOSIS: Methods in Parasitology: Preparing thin and thick blood films with

capillary or venous blood. Materials for finger: • Disinfectant • Swabs • Microscope slides (cleaned with

alcohol) • Sterile lancets • Special slide as spreader • Gloves

PREPARING THIN AND THICK BLOOD FILMS

Prepare the

microscope

slides

PREPARING THIN AND THICK BLOOD FILMS

(capillary blood)With the patient‘s left

hand,palm upwards, select thethird finger. (The big toecan be used with children.The thumb should never beused for adults or children).Use cotton wool lightlysoaked in alcohol to cleanthe finger. Let fingerair-dry.

PREPARING THIN AND THICK BLOOD FILMS

(capillary blood)

With a sterile

lancet

puncture the ball

of

the finger using a

quick rolling

action.

PREPARING THIN AND THICK BLOOD FILMS

By applying gentlepressure to the fingerexpress the first dropof blood and wipe itaway with dry cottonwool.Make sure no strandsof cotton remain onthe finger.

PREPARING THIN AND THICK BLOOD FILMS

Working quickly withcapillary blood andhandling clean slides onlyby the edges, collect theblood as follows:Apply gentle pressure tothe finger and collect asingle small drop of bloodabout the size ● on theend of the slide. This isfor thin film.

Venous blood can be used instead of capillary blood.

Use vacutainers with anticoagulant (EDTA)

For preparing thinand thick films use aglass capillary tube or automated pipette to drop theETDA blood.Do not use a plasticpipette!

PREPARING THIN AND THICK BLOOD FILMS

1. Hold the spreader at 30-45degree angle

against the surface of the slide. 2. Move the spreader back to touch the

drop of blood and allow the blood to extend along the edge of the

spreader. 3. Push the spreader with a steady hand

across the slide. 4. Air dry the film.

PREPARING THIN AND THICK BLOOD FILMS

Possible mistakes:

This is wrong!

This is correct!

PREPARING THIN AND THICK BLOOD FILMS

Observe right angle:

Angle too flat film too long

Angle too steep film too short

PREPARING THIN AND THICK BLOOD FILMS

Pressure on spreader: too strong

waves

Air bubble holes

PREPARING THIN AND THICK BLOOD FILMS

1. A large drop of blood is taken on the centre of a clean labeled slide ( a drop

of finger puncture or from well mixed

EDTA. tube of blood by capillary tube or

pipette). 2. With an other slide corner spread the drop over 1/2 an inch square area. 3. Air dry the film.

PREPARING THIN AND THICK BLOOD FILMS

Thick filmsCheck for the rightthickness:You should be ableto read thenewspaper!

PREPARING THIN AND THICK BLOOD FILMS

Additional mistakes

• Thick film too thick

• Thick film should be round!

PREPARING THIN AND THICK BLOOD FILMS

These slides look

o.k.

PREPARING THIN AND THICK BLOOD FILMS

FIXING OF THIN BLOOD FILMS:

* With absolute methanol, by immersing in a container of absolute methanol for 2 minutes.

* Methanol containing water must not be used to fix blood films.

PREPARING THIN AND THICK BLOOD FILMS

Staining thin blood film (Leishman stain): ☻Cover the blood film with undiluted

Leishman stain, but do not flood the slide, allow to stain for 2 min.

☻Add twice the volume buffered water. ☻Mix water and stain by bowling on the dilute

stain. ☻Allow to stain for 10 minutes.

☻Wash off the stain with tap water, wipe the back of the slide clean and stand it in a rack

for the smear to dry.

PREPARING THIN AND THICK BLOOD FILMS

 STAINING THICK BLOOD FILMS (Giemsa Staining):

  ☻Dilute the Giemsa stain as required: 10% solution for 10 minutes staining:

Measure 150 ml of buffered water in a 200 ml cylinder. Add 15 ml of Giemsa stain and mix

gently. ☻Cover the blood film Giemsa stain.

☻Allow to stain for 10 minutes . ☻Wash the stain using clean water.

☻ Wipe the back of the slide clean and stand it in a

rack for the smear to dry.

MICROSCOPIC EXAMINATION OF BLOOD

PLASMODIUM FALCIPARUM

PLASMODIUM VIVAX

MICROSCOPIC EXAMINATION OF BLOOD

PLASMODIUM MALARIAE

PLASMODIUM OVALE

MICROSCOPIC EXAMINATION OF BLOOD

For Malaria microscopic diagnosis we looking for:

1. Appearnce of RBC (characteristics)

2. Ring forms

3. Trophozoites

4. Schizonts

5. Gametocytes (macro & micro gametocyte)

PLASMODIUM VIVAX

RBC appearance:• Size: larger than mature RBC• Color: Pale• Shape: Round • Cytoplasmic inclusions: Schuffner’s

dots present• usually no more than one parasite is

observed within a single enlarged red blood cell

PLASMODIUM VIVAX

PLASMODIUM FALCIPARUM RBC appearance

• Size: Mature RBC• Color: Normal• Shape: Round • Cytoplasmic inclusions: Maurer’s clefts

may be present in late trophoziots

PLASMODIUM FALCIPARUM Ring Form Schizont stage

PLASMODIUM FALCIPARUM

Gametocytes of P. falciprum

Macro-gametocyte Microgametocyte

Size: larger than RBCShape: crescent –sharply pointed endCytoplasm: darkChromatine: compact mass near the centerPigment: black granules round the center.

Size: larger than RBCShape: kidney-bluntly round endCytoplasm: riddish Chromatine: fine granules scattered throughoutPigment: dark granules throughout

PLASMODIUM OVALE RBC appearance

Size: larger than mature RBC Color: pale Shape: oval Cytoplasmic inclusions: James’ dots

PLASMODIUM MALARIA RBC appearance

Size: smaller and older RBC Color: Normal Shape: Round Cytoplasmic inclusions: None

Thank you