blood film and staining

Vet. Heba Yasser

The blood film should be done immediately from fresh blood or within an hour of sampling in case of anticoagulated blood.

1. Two slides method: one of them is a spreader slide with its

two corner edges cut off.

N.B.You should wave the slide in the air to fasten drying because slow drying leads to movement of water out of erythrocytes leading to creantion.

Advantage and disadvantages>>>>>>>>>>>? H.W.

Place a small drop of well mixed blood near one end of the slide with applicator stick

2. Cover slip method:

Advantage and disadvantages>>>>>>>>>>>? H.W.

1. Free from holes caused by grease or dirt on the slide.

2. Not wavy film due to jerky movement during spreading.

3. Free from clear streaks resulted from chipped edge of the spreader slide.

4. Film is distributed in a single layer over a greater portion of the slide.

Too little blood

Grease and dirt holes on slide

Chipped spreader edge

Too much blood

Blood film must be fixed before staining to avoid being washed away during it.

Blood film is fixed using chemical fixatives which generate chemical bonds between proteins and other substances within the sample, increasing their rigidity e.g. methanol.

Neutral stain:

Several stains are used but all are based on methylene blue as a basic dye and is attracted to the nucleic acid while eosin is an acid and is attracted to the alkaline cytoplasmic proteins.

Wright’s stain:It is named for James Homer

Wright, who devised the stain, a modification of the Romanowsky stain, in 1902. Because it distinguishes easily between blood cells, it became widely used for performing differential white blood cell counts

Preparation:

1.5 g leishman powder dissolved in 1000ml methanol , filtered and keep for 3 months to ripen in brown bottles

Preparation:

Dissolve 800 mg of geimsa powder in 50 ml

glycerol and 50 ml ethanol, filter before use and kept in brown bottles

It is a water based neutral stain which consists of two solutions , it is a rapid method gives excellent results and useful in staining large no. of slides

Sol. A

Sol. B

D.W.

Neutral red: stain phagocytic structures. Pincyanol: stains mitochondria Peroxidase: azurophilic granules of

myloid leukemic cells. Brilliant cresyl blue (BCB), new

methylene blue (NMB): stains reticulocytes.

Reticulocytes are immature red blood cells, typically composing about 1% of the red cells in normal blood.

It develops and matures in the bone marrow and then circulate for about a day in the blood stream before developing into mature red blood cells.

They do not have a cell nucleus. They are called reticulocytes because of a reticular (mesh-like) network of ribosomal RNA that becomes visible under a microscope with certain stains such as new methylene blue.

H.W.

Reticulocytes stained with Wright stain

Stainned with NMB showing ribosomal RNA

Over staining due to prolonged time. Precipitating staining, due to deficient

washing, improper filtering of stain or evaporation of alcoholic stain.

Variation in staining in different areas of smear, due to acid or alkaline residues, improper draining of water.

Too blue slide and over stained granules due to acidic residues.

Pale blue and over stained eosinophil due to acidic residues

1. low power exam. To judge the quality of stain , cells distribution and presence of platelet clumps or microfilaria.

2. examine the cells for abnormalities. Finally starts a differential leucocytic

count.