Parasitology

EXAMINATION OF BLOOD Done 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 the third finger. (The big toe can be used with children. The thumb should never be used for adults or children). Use cotton wool lightly soaked in alcohol to clean the finger. Let finger air-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 gentle pressure to the finger express the first drop of blood and wipe it away with dry cotton wool. Make sure no strands of cotton remain on the finger.

Preparing thin and thick blood films Working quickly with capillary blood and handling clean slides only by the edges, collect the blood as follows: Apply gentle pressure to the finger and collect a single small drop of blood about the size ● on the end of the slide. This is for thin film.

 Venous

blood can be used instead of capillary blood.

 Use

vacutainers with anticoagulant (EDTA)

For preparing thin and thick films use a glass capillary tube or automated pipette to drop the ETDA blood. Do not use a plastic pipette!

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 films Check for the right thickness: You should be able to read the newspaper!

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: 2. Appearnce of RBC (characteristics) 3. Ring forms 4. Trophozoites 5. Schizonts 6. Gametocytes (macro & micro gametocyte)

 RBC

Plasmodium vivax 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

Size: larger than RBC Shape: crescent –sharply pointed end Cytoplasm: dark Chromatine: compact mass near the center Pigment: black granules round the center.

Microgametocyte

Size: larger than RBC Shape: kidney-bluntly round end Cytoplasm: riddish Chromatine: fine granules scattered throughout Pigment: dark granules throughout

Plasmodium ovale  RBC  Size:

appearance

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

Plasmodium malaria  RBC  Size:

appearance

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

Thank you