Hematology Chapter 6: Staining and Examination of Blood Films

Study Notes

Staining and Examination of Blood Films

Staining of blood films is a crucial step in hematology, allowing for the examination of cells and cell components.

History of Staining

Ehrlich introduced aniline dyes in the late 19th century, which were later modified by Romanowsky to create a stain that combines acidic and basic properties.
Romanowsky used a mixture of old methylene blue and eosin to stain the nucleus of a malarial parasite purple and the cytoplasm blue.

Romanowsky Stains

Commonly used Romanowsky dyes include Wright and Leishman stains.
Wright stain is a mixture of methylene azure and eosin.
Leishman stain is similar to Wright stain, but with a different preparation method.

Preparation of Romanowsky Stains

Wright stain is prepared by mixing methylene azure and eosin in a specific ratio.
Leishman stain is prepared by polychroming methylene blue with sodium carbonate, then mixing it with eosin B.

Appearance of Cells and Cell Components in Romanowsky-Stained Blood Films

Red cells appear pink with a central pale area.
Nuclei of leucocytes appear blue to purple.
Eosinophilic granules appear red-orange and distinctly visible.
Basophilic granules appear dark blue.
Platelets appear as violet granules.
Cytoplasm of monocytes appears gray-blue with fine reddish granules.
Cytoplasm of neutrophils appears light pink with lilac (pale purple) granules.
Cytoplasm of lymphocytes appears in varying shades of blue.
Malaria parasites appear with sky blue cytoplasm and red-purple chromatin.

Giemsa Stain

Giemsa stain is an alcohol-based Romanowsky stain that requires dilution in pH 7.1-7.2 buffered water.
It employs various azure compounds with eosin and methylene blue.
It is excellent in staining malaria parasites in thick films.

Microscopic Examination of Blood Films

Examine the film at low power (10x) to inspect for even distribution of cells, staining quality, and platelet clumping.
Survey the film at 10x magnification to get a general impression of its quality.
Identify areas where red cells are evenly distributed, just touching but not overlapping, and study their gross morphology at 40x.
Scan the film to get an impression of the quantitative distribution of white blood cells.
Identify any unusual or abnormal cells, and estimate the relative proportion of platelets.
Use the 100x objective for studying the fine details of cell morphology.

Staining Problems and Remedies

A stain that is too pale may indicate that the stain is too acidic due to a buffer problem.
To correct this, stain a second slide from the patient, increase the pH, and then assess for color of granules and red cell color.

Description

This chapter covers the principles and techniques of staining thin blood films in hematology, including the use of Romanowsky dyes. It also describes the appearance of cells and cell components in stained blood films.