Nada Mohamed Ahmed ,
MSC, MT (ASCP)i
Preparation of Blood Films
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Values:
To study morphology of RBC.
To study morphology of WBC.
To study morphology of Platelets.
To confirm diagnosis of blood cells disorders.
To examine hemoparasites(Malaria,
Trypanosoma, Babesia)
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Requirements:
1-Blood Sample:
-EDTA ant coagulated (venous whole blood).
-Finger prick blood (Capillary blood).
2- Stationary clean slide
3-Spreader slide
• Procedure:
• Use clean standard size glass slides (3 inch x 1
inch = 7.5 cm x 2.5 cm), wiped from dust just
immediately before use.
• Place a small drop of well mixed anticoagulated
whole blood, in the center line of the slide, about
1.5 to 2 cm from one end, with the aid of a
capillary tube.
• Immediately, without delay, with the aid of a
second clean slide with uniform smooth edges
(spreader slide), with a 30 –40 degrees angle,
move back so blood drop will spread along the
edge of the spreader slide, when this occurs,
spread, or smear move forward to make ideal .
Thin blood film consist of thick Head, body and
tail.
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Improper blood film
A: Blood film with jagged (with rough, sharp points )tail made
from a spreader with achipped end.
B: Film which is too thick
C: Film which is too long, too wide, uneven
thickness and made on a greasy slide.
D: A well-made blood film.
Examples of unacceptable smears
Examples of unacceptable smears
• Quality Control for blood film preparation :
• 1-Before preparing the films, you must
check that blood samples are free from clotsIf
clots are present the specimen is not used.
• 2-Films can be labeled with patient’s name and
/or Lab. No. on the thick end of the film itself,
after being dried, by using a pencil.
• 3-Thin blood film must be done immediately
as long as two to three hours after venous
blood collection.
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• principle of Romanosky stains
• Group of blood cells stains each
consists of Azure B (the basic
dye stains the acid portion of the
cell) and Eosin Y ( the acidic dye
stains the basic portion of the
cell).
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Romanovsky stains include:
1-Giemsa Stain
2-Wright’s Stain
3-Leishman Stain
• Leishman Stain:
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Requirements
Adequate air dried thin film
Buffer (PH 8.6) instead pure distilled water.
Procedure
1-Put the film on a staining trough rack.
2-Flood the slide with the stain.
3-After 2 minutes ( or more, if the stain in
newly prepared), add double volume of buffer or
distilled water, and mix the stain with water, until
a shiny layer is seen.
• 4- After 8 minutes, wash with a stream of tap
water.
• 5-Wipe the back of the slide with gauze.
• 6-Set the films in upright position on a dryer rack
then examine macroscopically and
microscopically.
Romanovsky Stain Blood Cells Characteristics
No. Cell Structure
Staining characteristics
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Red cells
Red or pinkish red
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Nuclei of all cell types
Purple/violet
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Lymphocyte cytoplasm
Blue
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Monocyte cytoplasm
Grayish
blue(or
glassy
gray)
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Platelets cytoplasm
Light blue
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Neutrophilic granules
Violet-pink
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Eosinophilic granules
Orange-red
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Basophilic granules
Purplish black/ Deep blue
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Platelets granules
Purple
Staining characteristics of a correctly stained normal
film:
Nuclei
Purple
Cytoplasm
 Erythrocytes
Deep pink
 Neutrophils
Orange-pink
 Lymphocytes
Blue; some small
lymphocytes
deep blue
 Monocytes
Grey-blue
 Basophils
Blue
Granules
 Neutrophils
Fine purple
 Eosinophils
Red-orange
 Basophils
Purple-black
 Monocytes
Fine reddish (azurophil)
Platelets
Purple
Eosinophilic granules
Blue nucleus
Basophilic granules
Staining faults
Too faint:
Staining time too short.
Excessive washing after staining.
Stain deposit:
Stain solution left inuncovered jar.
Stain solution not filtered.
Dirty slides.
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Other factors which affects the staining results
include :
1) Staining time,
2) pH of the staining solution, while
Sources Of Errors In Staining
Stain Precipitate or deposit: May obscure cell
details, and may cause confusion with inclusion
bodies. Eliminate by use of the stain before use.
pH of the buffer or water:
Too acidic pH causes too pinkish slides.
Too basic pH causes too bluish slides.
Improper stain timing may result in faded staining
or altered colors:
Too long staining time causes too blue slides
(overstaining).
Too short staining time causes too red slides.
Forced drying may alter color intensities and/or
distort cell morphology.
TOO ACIDIC
SUITABLE
TOO BASIC
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Non-stain related errors:
1-EDTA causes crenation of the cells after blood
collection.
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2-Severely anemic blood samples causes
slower drying (before staining) due to excessive
plasma.
3-Old blood specimens may cause
disintegration in WBC’s and decrease in their
numbers.
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4-Collection of blood in heparin causes blue
staining of RBC’s with bluish background,
which makes heparin unsatisfactory for
routine hematology testing, also heparin
induces platelet aggregation and clumping ,
with subsequent erroneous platelet count with
automated counters.