Blood Smear Preparation and Staining Techniques

• The laboratory activity covers the preparation, staining, and examination of blood smears.

Preparation of Blood Smears

• Manual and automated methods are available for preparing blood smears.

Manual Method

• Wedge smear utilizes two glass slides.
• The spreader's angle should be 25 degrees for Sieverds and 45 degrees for Merck.
• Beacom's method uses a glass slide and coverslip.
• Ehrlich's method utilizes two coverslips.
• Buffy coat smear is utilized when a patient's WBC count is less than 1.0 x 10^9/L.
• Thick blood smear is for examination of blood parasites such as malaria.

Automated Method

• Automated methods involve using a spun smear.
• This involves linear and centrifugal smears.

Blood Smear Advantages

• Blood smears can be properly labeled.
• They can be properly stored.
• They are easy to handle and prepare.

Ideal Blood Smear Properties

• Ideal blood smears should have a gradual transition from thick to thin.
• The smear should occupy 2/3 – 3/4 of the slide.
• It should be margin-free.
• It should have a feathered edge and rainbow appearance.
• There should be no overlapping of cells.
• The smear surface must be free from scratches, ridges, waves, and holes.

Ideal Blood Smear Specifications

• Glass slide size is 3 x 1 inch (75mm x 25mm).
• Drop of blood is 2 to 3 mm.
• Slide angle is 30 – 45 degrees.
• Size A should be 0.5 inches from the tip of the slide.
• Size B should be 0.25 inches or 1 cm from the end of the slide.
• Size C should cover 2/3 of the slide.

Drying Methods

• Blood smears can be dried by air drying.
• Additional drying methods are using a low flame, oven, or immersion in methyl alcohol.

Factors Affecting Smear Thickness

• The smear thickness is affected by the pressure of the spreader on the slide.
• The smear thickness is affected by the angle between the slide and spreader.
• The smear thickness is affected by the size of the blood drop and the speed of the spreader.

Additional Blood Smear Factors

• K2 EDTA is an ideal anticoagulant.
• Cells broaden during smear preparation, which affects the morphology of cells.
• A cold blast drier from about 30 cm can dry the smear.
• For high HCT, decrease the angle of the spreader.
• For low HCT, increase the angle of the spreader.
• EDTA preserves the blood in blood smears.
• Blood smears look at White Blood Cell differentiation.

Blood Smear Problems

• Too thick or too thin smears can be problematic.
• Hesitation bands can cause problems.
• Streaks or spots (areas of no blood) are issues.
• Narrow and thick smears are problematic.
• Crenation or incorrect staining creates issues for blood smears.
• Correct blood smears will look like a thumbprint.
• They should have a feathered edge and be of equal thickness.

Staining of Blood Smears

Staining Conditions

• Ideal pH in staining is 6.3 to 7.3.
• Buffer solutions include 0.05 M Sodium Phosphate (pH 6.4) and aged distilled water (pH 6.4 – 6.8).
• Sorensen's PBS can be used in Wright-Giemsa stain, with pH 7.2 when looking for malaria.
• Wash on the reverse side of the slide so as not to wash off basophil granules.
• May Grunwald Giemsa Stain is the preferred stain.

Romanowsky Stain

• Romanowsky stain components are polychromatic in nature.
• Methylene Blue or Azure B are components.
• Eosin Y is another component.
• Examples include Wright stain (US), Leishman stain (Europe), and Giemsa stain (Europe).
• Wright Stain Solution contains Wright Stain Powder (3g), Glycerin (Stabilizer) (30 mL), and Absolute Methyl Alcohol (Fixative) (1000mL).

Panoptic Stain

• Panoptic stain is a combination of 2 or 3 stains.
• Panoptic fast staining technique: hematological, differential, non-vital staining for dead cells.
• This staining technique is based on May Grünwald-Giemsa's traditional Romanowsky type staining.
• Procedure is based on immersions.
• Examples include May Grunwald-Giemsa, Jenner-Giemsa, and May Grumwald-Wright.

Wright Stain Reactions

• Neutrophils have a dark purple nucleus and fine lilac pink granules in the cytoplasm.
• Eosinophils have a dark blue nucleus and red to orange granules in the cytoplasm.
• Basophils have a dark blue nucleus and dark blue to blue black granules in the cytoplasm.
• Lymphocytes have a dark purple nucleus and sky blue cytoplasm.
• Monocytes have a lighter purple nucleus and pale blue to gray-blue cytoplasm with fine reddish granules (ground glass appearance).
• Platelets have lilac blue.
• Red blood cells have salmon pink.
• Reticulocytes have pinkish gray.

Wright Stain Procedure

• Cover the smear in Wright's stain, then dilute with an equal volume of buffered water, and wash.

Acidic Wright's Stain Issues

• Insufficient staining time can cause the stain to become too acidic.
• Prolonged buffering or washing can cause the stain to become too acidic.
• Exposure of stain or buffer to acid fumes makes the stain too acidic.
• Old stain with methyl alcohol oxidized to formic acid will be too acidic.
• Remedies include prolonging the staining time.
• Another fix is to check the pH of stain and buffer.
• Correct with alkali if needed as well.
• Shorten the buffering time.

Alkaline Wright's Stain Issues

• Thick blood smears, prolonged staining, or insufficient washing can lead to alkaline stains.
• An alkaline pH of stain, buffer, or water can cause issues.
• Using a new stain solution which has not stood can cause issues.
• Stain that is too basic can cause the stain to be too alkaline.
• Solutions are to check pH of stain and buffer water.
• Shorten staining time, prolong buffering time, and check the incubation time of the stain.

Examination of Blood Smears

• Several methods for scanning smears include two field mender, four field mender, crenellation technique/battlement track scan pattern, exaggerated battlement method, and strip method.
• Only one method should be used in scanning one smear to avoid counting the same cell twice or more.

White Blood Cells

• White blood cells can be classified further, some examples follow.

Neutrophils

• Neutrophils are the most abundant type of white blood cell.
• They make up 7-30% of nucleated cells in the bone marrow.
• They have 2-5 nuclear lobes connected by threadlike filaments.
• (Segmented neutrophils).
• Their half-life is 7 hours.
• They function in phagocytosis and destruction of foreign materials or organisms that are not antigen-specific.

Eosinophils

• Eosinophils: Cytoplasm contains refractile, orange-red secondary granules.
• They have a bilobed nucleus.
• Their half-life is 18 hrs before entering the tissue, where they survive for at least 6 days.
• They function in Phagocytosis and modulation of inflammatory response.
• Eosinophils leave the blood when adrenal corticosteroid hormones increase.
• They destroy helminths by generating potent oxidants and releasing cationic proteins.
• They react to allergic reactions.

Basophils

• Basophils: Cytoplasm is colorless and contains a large number of large blue-black granules.
• They have a lobulated nucleus.
• Their chromatin pattern is clumped.
• As the cell matures, the granules become more metachromatic (red-purple) because of increasing acid mucopolysaccharide (heparin) content.
• They function in hypersensitivity reactions.
• Along with eosinophils, they are involved in the control of helminth infections.
• They contribute to worm expulsion.

Monocytes

• Monocyte size: 15-20 um (larger than neutrophil).
• Monocytes remain in the circulation for approximately 30 hours.
• They have a ground-glass appearance - Blue-Gray with fine azure granules (AZURE DUST).
• There may be cytoplasmic pseudopods or blebs
• N:C Ratio is 1
• They have a deeply indented (horseshoe shaped) nucleus.
• Nucleoli: None

Lymphocytes

• Small Lymphocyte: 8-10 um
• Medium Lymphocyte: 10-12 um
• Large Lymphocyte: 12-16 um
• Large lymphocytes are also known as “LARGE GRANULAR LYMPHOCYTE,” believed to be NK cells.
• T LYMPHOCYTE: 60-80% = Long lived (4-10 years)
• B LYMPHOCYTE: (10-20%) = Short lived (3-4 days)
• NULL LMPHOCYTE : (10% lymphoid population)

Identification

• Cell Size and Nucleus: Cytoplasm Ratio help with Identification.
• HIGH RATIO – increased nucleus in relation to cytoplasm; YOUNG CELLS helps identify cells.
• LOW RATIO – decreased nucleus in relation to cytoplasm; MATURE CELLS helps identify cells.
• Cytoplasm Characteristics for cells include color of the background of the cytoplasm.
• Cytoplasm Characteristics include presence or absence of granules and color and size of granules.
• Nuclear Characteristics for cells include Shape, Color, and Chromatin.
• Nuclear Characteristics for cells includes presence or absence of nucleoli.