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5 - Blood Film Preparation, Staining, and RBCs Manual Counting-2

This document outlines the procedures for blood film preparation, staining, and manual counting of red blood cells (RBCs). It describes the types of blood films, the wedge smear preparation technique, and the staining methods using Romanowsky stains. Additionally, it includes details on manual RBC counting and the normal ranges for RBC counts in men and women.

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10 views25 pages

5 - Blood Film Preparation, Staining, and RBCs Manual Counting-2

This document outlines the procedures for blood film preparation, staining, and manual counting of red blood cells (RBCs). It describes the types of blood films, the wedge smear preparation technique, and the staining methods using Romanowsky stains. Additionally, it includes details on manual RBC counting and the normal ranges for RBC counts in men and women.

Uploaded by

tanyeol1512
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© © All Rights Reserved
We take content rights seriously. If you suspect this is your content, claim it here.
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CLS 291 Clinical Hematology 1

Lecture 5
Blood Film Preparation, Staining, and
RBCs Manual Counting
Outlines

I. Making blood film


II. Staining Blood Films
III. Red blood cell manual counting
I. Blood Film - Wedge Smear
Preparation
Types of blood film
● The aim of blood film allows the evaluation of white blood cells (WBCs, leucocytes), red blood
cells (RBCs, erythrocytes), and platelets (thrombocytes) size and morphology.

● Staining of blood film helps in differentiating cells and detecting the presence of abnormality
in microscopic examinations of blood and bone marrow samples.
● There are two types of blood films:

● Thick blood film


● Allow a more efficient detection of parasites.
(increased sensitivity 11 times than thin
smear).
● No Fixation is used.

● Thin blood film


● Used for examining Blood Cell morphology.
● Fixed smear.
Wedge Smear Preparation
● Prepare the blood film as follows:
Wedge Smear Procedure
Prepare the blood film as follows:

1. Use 2 clean glass slides. (one for spreading and one for the blood
film). (A)
2. Label the slide with the patient's MRN# and your initial.
3. Add a small drop of EDTA blood sample about 3 mm in diameter
placed at one end of the slide.
(B)
4. Holds the pusher slide securely in front of the drop of blood at a 30-
to 45-degree angle to the smear slide (A).
5. The pusher slide is pulled back into the drop of blood and held in
that position until the blood spreads across the width of the slide (B). (C)
6. Then quickly and smoothly pushed forward to the end of the smear
slide, creating a wedge smear (C).

https://www.youtube.com/watch?v=rqXy45sRJkw
https://www.youtube.com/watch?v=nbRUiWl2Qrs Well-made blood smear
Unacceptable Blood Smear

In a well-made when
the slide is held up to
the light, the feather
edge of the smear
should have a
“rainbow” appearance.
II. Staining Blood Films
Staining of blood film
● Types of staining: Romanowsky stains (the routine stain in hematology).

● Romanowsky stains include a number of stains, such as:

1. Jenner
2. Giemsa

3. Leishman’s stain

4. Wright’s stain
Unstained vs Stained Blood Film

Unstained Blood Film Stained Blood Film


What colors do you see?
Acidic and basic staining mechanisms
● It depends on two components:

● Acidic dye (eosin Y):

● Binds to basic parts of the


cell as cytoplasm and Hb
and gives them red color.

● Basic dye (azure B):

● Binds to acidic parts of the


cell as the nucleus and Acid and basic dyeing are seen as ion exchange processes. Mobile counterions,
shown as Na+ and Clˉ, in actuality, depend on the species present in the
gives them blue color. staining solution.
Acidic and basic staining mechanisms
pH of Cell Component Color after staining with Romanowsky stains

Hb (basic-cationic) Red
by Eosin Y (acid-anionic)
Nucleus (acid-anionic) Blue–purple
Due to the negative charge of phosphate by azure B (basic-cationic)
groups of nucleic acids.
Basophilic granules (acid-anionic) Dark blue
by azure B (basic-cationic)
Eosinophilic granules (basic-cationic) Red
by Eosin Y (acid-anionic)
Nutrophil granules Purple
mixed of both Eosin Y and azure B
Blood cell appearance after staining

Basophil has heparin in its granules Eosinophil granules are basic. Thus,
which is acidic. Thus, the azure B dye eosin Y (acidic dye) of the stain will
(basic dye) of the stain will bind to the bind to basic granules and gives
granules of basophil and stain it blue. them red color.
Blood cell appearance after staining
A well-made stained peripheral blood smear has the following
characteristics:

1. The red blood cells (RBCs) should be pink to salmon.


2. Nuclei are dark blue to purple.
3. Cytoplasmic granules of neutrophils are lavender to lilac.
4. Cytoplasmic granules of basophils are dark blue to black.
5. Cytoplasmic granules of eosinophils are red to orange.
6. The area between the cells (background) should be
colorless, clean, and free of precipitated stains.
Blood Film Staining Procedure
● Procedure steps

I. Blood Smear Preparation: Wedge Smear.

II. Fixation: [(physical fixation) by Air drying the smear]. Don’t forget to label the slide.

III. Staining: [(chemical fixation) by Methanol, staining, and washing].

IV. Microscopy: Examine your slide under the microscope.


Blood Film Staining Procedure
III. Staining procedure: (Leishman’s stain):
1. Immerse the slide for 2 minutes in 100% Methanol (fixation step).

2. Immerse the slide for 4 minutes in a solution containing: 100% Leishman’s stain.

3. Immerse the slide in the first 100% Buffer (washing step) by immersing the slide for 1 minute.

4. Wash the slide in the second 100% Buffer (washing step) by immersing the slide 5 times.

5. Wash with water (carefully).

6. Air dry, then examine under the microscope.


Microscopic Examination
1. Examine under the microscope.
2. Start with low power (X10) to find the suitable area of ideal thickness where the
cells are appropriately separated.
3. Change to (X40) to see the morphology. <<< Most of your time, you will use this magnification.
4. Change to (X100) to see inclusions (if present) – (use immersion oil only with 100x)

(X100)
Different areas of thickness found in a smear

Body of smear – have too much Rouleaux Monolayer - ideal area for investigation Edge of smear – less cells
Microscopic Examination

Use the (zig-zag motion) scanning technique for examining your blood smear on the 40X objective lens.
II. Red Blood Cells Counting
Manual counting of RBCs
Manual counting of RBCs
Manual counting of RBCs
Manual counting of RBCs
● In manual RBC counting, we are counting the number of RBCs in 1 µl.

Normal range of red blood cell count:

● Men 5.0 +/- 0.5 x 10^12/L


● Women 4.3 +/- 0.5 x 10^12/L

[x 10^12/L] is a unit. Don’t use it in any of your calculations!

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