Blood Smear Preparation for Malaria Microscopy

							                  1 Basic Lab Tech




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    Preparation of Blood Smear
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      for Malaria Microscopy
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                                     SantiagoA/LuchavezJ/2002
                              2 Basic Lab Tech


    A THICK AND A THIN BLOOD FILM ON THE SAME SLIDE
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                                                 SantiagoA/LuchavezJ/2002
                                          3 Basic Lab Tech

             BLOOD SMEAR FOR MALARIA
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            Thick Film                              Thin Film
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I   •   Layer of red blood cells      •   Single layer of red blood
        10-20 times thicker than in       cells
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        a thin film                   •    Used as label to identify
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        cell ghosts (staining
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                                      •    Used to identify parasite
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        unfixed red cells causes          species, after they have
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        hemolysis)                        been seen in the thick
        Used to detect parasites          film
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        and estimate parasite         •    Gives specificity to
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        density                           diagnosis
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    •   Gives sensitivity to
        diagnosis

                                                                SantiagoA/LuchavezJ/2002
                                              4 Basic Lab Tech

      PREPARATION OF BLOOD SMEAR FOR MALARIA
      STEP 1
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M   1. Clean the 4th finger from the thumb with cotton wool dampened
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        with 70% alcohol, using firm strokes to remove dirt and grease
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        from the finger. Air-dry the finger. Using a sterile lancet, puncture
        the ball of the patient’s finger. Apply gentle pressure to the finger
        to express the first drop of blood and wipe it away with dry cotton.

                                                                    SantiagoA/LuchavezJ/2002
                                       5 Basic Lab Tech
   PREPARATION OF BLOOD SMEAR FOR MALARIA
   STEP 2




2. Apply gentle pressure to the finger then collect 3 small drops of
   blood on one side of a clean slide and 1 small drop next to the
   3 drops, leaving some space between the thick and thin smears
   to be made. Handle clean slides only by the edges. Wipe the
   remaining blood away from the patient’s finger with dry, clean
   cotton.
                                            6 Basic Lab Tech

     PREPARATION OF BLOOD SMEAR FOR MALARIA
     STEP 3-4
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    3. Make the thin smear first. Place slide on a flat, firm surface.
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       Bring down a second slide (spreader) on the 1 small drop at
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       an angle of 45o, allow the blood to run along its edge.

    4. Firmly push the angled slide away from the blood towards the
       other end of the slide.
                                                                SantiagoA/LuchavezJ/2002
                                     7 Basic Lab Tech
PREPARATION OF BLOOD SMEAR FOR MALARIA
STEP 5




5. After spreading the thin smear, make the thick smear. With
   the corner of the second slide, quickly join the drops of
   blood, and spread them in a circular motion until it is about
   one centimeter in diameter. The thickness should be such
   that it is just possible to see news print through it.
                                     8 Basic Lab Tech
PREPARATION OF BLOOD SMEAR FOR MALARIA
STEP 6


                                             Label here




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6. Using a lead pencil, label the smear with the name or
   number of the patient and date on the thick portion of the
   thin film. Air-dry slide on a flat, level position, protected
   from insects, dust and extreme heat.
                                      9 Basic Lab Tech
PREPARATION OF BLOOD SMEAR FOR MALARIA
STEP 7




7. Fix the thin smear with methanol by using a dropper or by
   dipping it in the solution for a few seconds. Air dry the smear.
   Do not fix the thick smear.
                                     10 Basic Lab Tech
 PREPARATION OF BLOOD SMEAR FOR MALARIA
 STEP 8




8. Stain thick and thin smears with Giemsa or Wright’s stain for 10-
   15 minutes then wash under running tap water.
   (10% Giemsa stain preparation: 1 part stain + 9 parts buffered
   water)
                                       11 Basic Lab Tech
PREPARATION OF BLOOD SMEAR FOR MALARIA
STEP 9




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 9. Air-dry smears in a vertical position then examine under
    oil immersion.
                                        12 Basic Lab Tech

    COMMON FAULTS IN BLOOD SMEAR PREPARATION
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    Too big thin smear, thick smear   Edge of spreader slide chipped
A   badly positioned
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                 Blood smears spread on a greasy slide
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2       Too much blood                              Too little blood
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                                                                  SantiagoA/LuchavezJ/2002
                              13 Basic Lab Tech




              Acknowledgements:

National Center for Infectious Diseases, DOH

Japan International Cooperation Agency

US-NAMRU 2 Malaria Project

WHO Roll Back Malaria Project

Global Fund Malaria Project