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
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