With Procedures, Combined Thick and Thin Films/Smears

• Combined Thick and Thin Films/Smears

  • The thick smear is first dehemoglobinized and the two are then stained together.

  • Do not allow the methanol to contact the thick film when fixing the thin film

  • The stained smear is examined first. If the thin smear is negative, the thick smear should be searched for parasites.
• Combined Thick and Thin Films/Smears

  Preparation of Thin and Thick Smear

  = Materials

  + Blood samples

  + 70% Isopropyl alcohol

  + Clean Glass slides

  + Marking pen

  + Cotton

  + Lancet
• Combined Thick and Thin Films/Smears

  = Thin Blood Smear

  • Made in same manner as that used for a differential count in Hematology.

  • The suggested “Push Slide Technique” or “Wedge Method” assures that the thin area of the slide has one layer of evenly distributed cells.

  • For best results, slides should be alcohol-cleaned, grease free, and thoroughly clean.
• + Thin Blood Smear

  = Procedure:

  • Place a small drop of blood near one end of an alcohol-cleaned microscope slide.

  • Using a second clean glass slide as a “Push slide” or “Spreader”, back spreader into the blood at a 30-degree angle

  • In one continuous movement, draw the blood across the slide. A properly made smear will have a thick end and a thin end that tapers to a feathery edge. Most of the area of slide will be a thin layer one cell thick.
• Combined Thick and Thin Films/Smears

  = Thick Blood Smear

  = Procedure

  • Place 2-3 small drops of whole blood close together at one end of an alcohol-cleaned glass slide.

  • With one corner of another clean glass slide. Mix the drops together in a circular motion over an area of 2 cm in diameter (About size of a nickel). If the slide has not been properly cleaned or the blood is too thick. The blood may flake off the slide during staining.
• + Thick Blood Smear

  = Procedure

  • Mixing should be continuous for at least 30 seconds to prevent formation of fibrin strands (If anticoagulated blood is used, this step should be eliminated).

  • Slides should be allowed to air dry thoroughly at room temperature. Do not heat.

  • Dry slides should be laked to remove hemoglobin. This can be accomplished by placing the slides in buffer solution prior to staining or in Giemsa solution itself.
• + Thick Blood Smear

  Note: if slides are not stained immediately, they should be laked in buffer prior to storage. As time passes. The removal of hemoglobin will be more difficult.
• Wright’s Staining

  = Principle

  • Wright stain is a permanent stain for blood smears that contains fixative (Alcohol) and stain one solution. Smears do not require fixation before staining.

  Materials and Reagents

  + Blood samples

  + Clean, Dry glass slides

  + Wright’s stain

  + Distilled water

  + Microscope

  + Staining Rack

  + Phosphate buffered water.
• Wright’s Staining

  Procedure:

  • Place slide on-level staining rack and cover the surface with commercially prepared Wright’s stain. Let them stand for 1-3 minutes.

  • Add equal volume of phosphate-buffered water to the slide. Mix by gently blowing on the surface of the fluid.

  • After 4-8 minutes, flood the slide with phosphate buffer, rinsing off the stain.

  • With a paper towel, wipe off the excess stain from the bottom of the slide and stand upright position to air dry.
• Wright’s Staining

  = Procedure for Thick Smear:

  • Before staining, thick blood films should be laked in distilled water to lyse the red cells and then air-dried.

  • Staining procedure is similar for thin smears.
• Giemsa Staining

  = Principle

  • The Giemsa stain is a permanent stain for blood smears that contains a separate fixative (Methanol) and stain solutions.
• Giemsa Staining

  = Procedure on Thin Smears

  • Fix blood films in absolute methanol (Acetone-free) for 30 seconds.

  • Allow slide to air-dry at RT.

  • Prepare a solution 1 part Giemsa stock to 10-50 parts buffer (pH 7.0-7.2). Immerse slide in stain for 10-60 seconds.

  • Dip slides briefly in phosphate buffered water or rinse with tap water. With a proper towel. Wipe off the excess stain from the bottom of the slide.

  • Drain thoroughly in vertical position and allow to air-dry
• Giemsa Staining

  = Procedure on Thick Smears

  • A greater volume of blood can be examined for parasitic forms when using thick smears, but the organisms may lack morphologic definition needed for species identification.

  • Perform same procedure as for thin smears starting at step 3.

  • Lack of methanol fixation allows for lysis or red cells in Giemsa staining solution
• INTERPRETATION STAINING RESULTS

  = Components

  • Erythrocytes

  = Wright's stain

  • Light Tan, Reddish or Buff

  = Giemsa Stain

  • Pale Red
• INTERPRETATION STAINING RESULTS ( WBCs )

  = Components

  • Nuclei

  = Wright's stain

  • Bright Blue

  = Giemsa Stain

  • Purple
• INTERPRETATION STAINING RESULTS ( WBCs )

  = Components

  • Cytoplasm

  = Wright's stain

  • Light Blue

  = Giemsa Stain

  • Pale Purple
• INTERPRETATION STAINING RESULTS ( WBCs )

  = Components

  • Eosinophilic Granules

  = Wright's stain

  • Bright Red

  = Giemsa Stain

  • Deep Pink to Purple
• EXPECTED STAINING RESULTS ( INTRAERYTHROCYTIC PARASITE )

  = Component

  • Cytoplasm

  = Wright's Stain

  • Pale Blue

  = Giemsa Stain

  • Blue
• EXPECTED STAINING RESULTS ( INTRAERYTHROCYTIC PARASITE )

  = Component

  • Nuclear Membrane

  = Wright's Stain

  • Red

  = Giemsa Stain

  • Red to Purple-red
• EXPECTED STAINING RESULTS ( INTRAERYTHROCYTIC PARASITE )

  = Component

  • Inclusions/dots

  = Wright's Stain

  • Stain Poorly

  = Giemsa Stain

  • Red
• EXPECTED STAINING RESULTS ( MICROFILARIAE )

  = Component

  • Sheath

  = Wright's Stain

  • Stain Poorly

  = Giemsa Stain

  • Stain Poorly
• EXPECTED STAINING RESULTS ( MICROFILARIAE )

  = Component

  • Nuclei

  = Wright's Stain

  • Pale to Dark Blue

  = Giemsa Stain

  • Blue to Purple