Preparing a blood smears is crucial methods in hematology, vital for the microscopic evaluation of blood cells. These steps enable a comprehensive analysis of component of ( C.B.C) complete blood count like red blood cells (RBCs), white blood cells (WBCs), and platelets, supporting the diagnosis and tracking of different hematological conditions.

Importance of Blood Smears

Blood smears play an important role in hematology diagnostics, complementing automated cell counters by facilitating a thorough visual analysis of blood cells. Their significance includes:

• Morphological Evaluation – Assists in recognizing atypical cell shapes, sizes, and inclusions that may suggest conditions such as anemia, leukemia, or infections.
• Identification of Blood Parasites – Essential for the diagnosis of malaria, babesiosis, and other parasitic diseases.
  Recognition of Immature and Abnormal Cells – Important for spotting blasts, reactive lymphocytes, and other unusual cell types that automated analyzers might misidentify.
• Validation of Automated Results – Blood smears act as a confirmation method when automated devices highlight abnormal findings.
• Affordable and Accessible – In contrast to costly automated equipment, manual blood smear analysis is economical and readily available, particularly in resource-limited environments.

Types of Blood Smear

1. Thin blood film ( unlysed fixed sheet of individual blood cells )
2. Thick blood film ( a multilayered film of unfixed lysed blood )

Preparation of blood smear

Thin Blood Film

Thin PBF can be prepared from anticoagulated blood obtained by venipuncture or from free flowing finger prick  blood by any of the following three techniques :

1. Slide Method

Procedure:

• Place a drop of blood in the center of a clean glass  slide 1 to 2 cm from one end.
• Place another slide (spreader) with smooth edge at an angle of 30-45⁰ near the drop of blood.
• Move the spreader backward so that it makes contact with  drop of blood.
• Then move the spreader forward rapidly over the slide.
• A thin peripheral blood film is thus prepared
• Dry it and stain it.

2. Cover Glass Method

Procedure:

• Take a clean cover glass.
• Touch it on to the drop of a blood.
• Place it on another similar cover glass in crosswise direction with side containing drop of blood facing down.
• Pull the cover glass quickly.
• Dry it and stain it.

3. Spin Method

Procedure:

• This is an automated method.
• Place a drop of blood in the center of a glass slide.
• Spin at a high speed in a special centrifuge, cytospin.
• Blood spreads uniformly.
• Dry it and stain it.

Thick Blood Film

Procedure:

• Place a large drop of blood in the center of a clean glass slide.
• Spread it in a circular area of 1.5 cm with the help of a stick or end of another glass slide.
• Dry it
• Staning

The shape of blood film

Qualities of a Good Blood Film

A quality blood smear is crucial for precise microscopic analysis, especially in hematology and parasitology. Below are the essential characteristics of a properly prepared blood smear:

1. Appropriate Thickness (Feathered Edge)
   The smear should be thin enough to observe individual cells without overlap. The feathered edge must exhibit a smooth gradient, tapering towards the end.
2. Uniform Cell Distribution
   Red blood cells (RBCs) should be evenly dispersed without any clumping. White blood cells (WBCs) ought to be distributed uniformly rather than being clustered at the edges. Platelets must remain unaggregated.
3. Correct Size and Length
   The smear should cover about two-thirds to three-fourths of the slide. It should feature a well-defined feathered edge with no sudden stops or streaks.
4. Smooth and Uniform Appearance
   The smear should be free of ridges, holes, or any irregularities. It should not be excessively thick or too thin.
5. Absence of Streaks or Lines
   Streaks may occur due to uneven spreading speed, dirt on the slide, or an incorrect angle while spreading.
6. Adequate Staining Quality
   Staining (such as Wright, Giemsa, or Leishman stain) should be consistent, avoiding both overstaining and understanding. WBCs should have clearly visible nuclei. RBCs must appear light pink, and platelets should be distinctly visible.
7. No Artifacts or Contaminants
   There should be no water artifacts, dust, or residues from the stain. Cellular structures must not show distortion from inadequate drying or improper stain preparation.
8. Adequate Drying
   The smear must be allowed to air dry quickly to avoid artifacts and maintain cell integrity.

Parts of a Thin Blood Film

A peripheral blood film consists of 3 parts

1. Head i.e. the portion of blood film near the drop of blood.
2. Body i.e. the main part of the blood film.
3. Tail i.e. the tapering end of the blood film.

Common cause of a poor blood smear

1. Drop of blood too large or too small
2. Spreader slide pushed across the slide in a jerky manner
3. Failure in keep the entire edge of the spreader slide against  the slide while making the smear
4. Failure in keep the spreader slide at a 30⁰ angel with the slide
5. Failure to push the spreader slide completely across the  slide
6. Irregular spread with ridges and long tail: edges of spreader dirty or chipped ; dusty slide
7. Holes in film – slide contaminated with fat or grease and air  bubbles
8. Cellular degenerative changes: delay in fixing inadequate fixing time or methanol contaminated with water

Examples of unacceptable smears

• Blood  film  with  jagged  tail  made  from  a  spreader  with  a chipped end.
• Film which is too thick
• Film  which  is  too  long,  too  wide,  uneven  thickness  and  made on a greasy slide.
• A well-made blood film.

Biologic causes of a poor smear :

• Cold agglutinin – RBCs will clump together. Warm the blood at 37° C for 5 minutes, and then  remake the smear.
• Lipemia – holes will appear in the smear. There is nothing you can do to correct this.
• Rouleaux – RBC’s will form into stacks resembling coins. There is nothing you can do to correct this.

Fixation of blood smear

Fixing a blood smear involves preserving the structure of blood cells to prevent deterioration and maintain cellular integrity for microscopic analysis. This step is crucial for preparing blood smears for staining, which is frequently utilized in hematology and pathology.

It  is  important  to  prevent  contact  with  water  before fixation is complete. Methyl alcohol (methanol) is the choice, although ethyl  alcohol (“absolute alcohol”) can be used. Methylated spirit (95% ethanol) must not be used as it  contains water.

Types of Fixation and Their Procedures

• Methanol Fixation (Most Common)
  Procedure:
  • Allow the blood smear to dry in the open air completely.
  • Submerge the slide in a container filled with absolute methanol for 2–3 minutes.
  • Take it out and let it dry before applying the stain (e.g., Wright’s or Giemsa stain).
  • Purpose: Maintains cell structure by stopping lysis and dehydration.
• Heat Fixation (Less Common for Blood Smears)
  Procedure:
  • Let the smear dry in the open air completely.
  • Quickly pass the slide through the flame of a Bunsen burner 2–3 times.
  • Allow it to cool before staining.
  • Purpose: Primarily used in microbiology for fixing bacterial samples but less frequently for blood smears due to possible distortion of cells.
• Formalin Vapor Fixation (Rarely Used)
  Procedure:
  • Position the air-dried slide containing the blood smear inside a closed container with a few drops of 37% formalin.
  • Allow it to fix for 10–15 minutes.
  • Take the slide out and let it air before staining.
  • Purpose: Employed in specific staining methods when alcohol fixation is not appropriate.
• Acetone Fixation (For Special Stains)
  Procedure:
  • Immerse the dried smear in acetone for 1–2 minutes.
  • Remove it and let it dry in the open air before staining.
  • Purpose: Utilized for fixing smears when testing for particular components such as lipids or enzymes.