Preparation and Staining of Peripheral Blood Films Flashcards
Peripheral blood films can be made directly from
Skin puncture
Tube of EDTA-anticoagulated venous blood
Best choice for PBS
Anticoagulant-free blood
With a skin puncture, the phlebotomist must remember to
wipe away the first drop of blood
Some patients’ blood undergoes an in vitro phenomenon called __________ when anticoagulated with EDTA.
platelet satellitosis
Describe platelet satellitosis
The platelets surround or adhere to neutrophils
When platelet adhere to neutrophils it potentially causes
Pseudothrombocytopenia
Can result from EDTA induced platelet clumping
Low platelet counts
Falsely increased WBC count (pseudoleukocytosis)
Manual Blood Film Preperation
Coverslip method
Wedge type
grease free, square coverslip 22 sq.mm.
superior WBC distribution
Coverslip method
Coverslip method should use
Use of two 1x3 in glass slides
Demonstrate coverslip method
A drop of blood placed on the center of the lower slide,
then the top slide is placed on the blood drop so that the ends of the 2 slides overlap slighty. Then the 2 slides are pulled apart in a smooth, parallel motion.
Variations of the ______ are the most widely used for smear preparation.
wedge technique
Wedge type smear is termed as
Wedge, spreader-slide or push wedge
Automated Blood Film Preparation
Wedge (Push) Type
Centrifugal (Spinenr) Method
Automated Slide Makers/Stainers
Portable semi-automatic instrument that simulates the manual spreader-type technique
Wedge (Push) Type
Automated instruments that utilize centrifugation to spread a monolayer of whole blood on a 1x3 inch glass slide
Centrifugal (Spinner) Method
Smear preparation, stain, differential count report, platelet estimate, captures digital image, barcoding
Automated Slide Makers/Stainers
The film is _____________________ the length of the slide
two thirds to three fourths
The film is ___________, very slightly rounded at the
___________, not bullet shaped
o This provides the widest area for examination.
finger shaped
feather edge
The ________ of the film are visible.
lateral edges
The film is smooth with a gradual transition from
Thin to thick areas
The film is smooth without irregularities,
no waves, no holes, or streaks and no bubbles.
When the slide is held up to the light, the thin portion
(feather edge) of the film has a
“rainbow” appearance.
It should allow at least ___ low power fields in which ____ of
the RBCs do not overlap.
10, 50%
How do we know that it is not the area for differential count?
Rouleaux formation is the linking of RBCs into chains resembling stacks of coins
best area for starting differential leukocyte count
Monolayer (middle part)
What is differential leukocyte count?
100 WBCs to be differentiated
UNACCEPTABLE PERIPHERAL BLOOD FILMS
Chipped, rough edge
Hesitation in forward motion
Drop of blood too small
Dirt or grease on the slide (elevated lipids in blood specimen)
The faster the smear
The shorter the smear
More amount of blood in smear will produce
A thicker longer smear
Widely used in diagnosis of blood parasites, particularly malaria (10x thickness of normal smears)
Thick Blood Smear
Accepted thickness of thick blood smear
If you are able to read through the fine print of the newspaper
Stains for thick blood smear
Giemsa Stain
Field’s Stain
Stain for both thin and thick smears to examine blood for malarial parasite
Giemsa Stain
What is it that you should not forget about giemsa? What makes it different from Wright’s when all are actually Romanowksy stain?
prepared by diluting Giemsa stain powder in a buffered bacteria., Giemsa is used for biological specimen like bacteria and parasite. Wrights Stain is used for non-blood specimen
Histological method for staining thick blood smears for malarial parasites.
Field’s Stain
corresponds to a drop of blood spread over a circle approx.
2 cm in diameter
The blood smears are mostly done for
Differential Leukocyte Count
Essentially all specimens received for routine testing in the hematology section of the laboratory have been collected in _________topped tubes.
Lavender (purple) topped tubes.
Lavender topped tubes contain
Ethylene-diaminetetraacetic acid (EDTA)
EDTA anti-coagulates the blood by
Chelating calcium that is essential for coagulation
_______________ is often preferred to the powdered form because it mixes more easily with blood.
Liquid tripotassium EDTA
why do we need to preserve the morphology of the RBCs and the amount of the analytes at the time of collection
To prevent unwanted changes that occurs when blood is collected from the body.
And in order for it to represent the exact conditions of the specimen at the time of collection
Fixation and staining of smears is done using the ff.
3-5 mins absolute methyl or ethyl alcohol
1-2 mins formalin solution in 50% alcohol
Principle of Staining
Acidic dyes unite with the basic components of the cell (cytoplasm) and conversely basic stains/dyes are attracted to and combine with the acidic parts of the nucleus (e.g. nucleic acids)
First to use simple aniline dyes, first in sequence then pre-
mixed acidic-basic stains (neutral dyes) = triacid: orange g, acid fuchsin, methyl green
Ehrlich
The precipitate formed after mixing eosin and methylene
blue has combined staining properties of the parent
dyestuffs
Jenner (1889)
He said that this precipitate that formed has improved
staining capacity
Jenner (1889)
Defined as a stain containing MB (methylene blue) and/or
its products of oxidation and a halogenated fluorescein dye
usually eosin;
Romanowsky (1890)
The polychromed methylene blue has wider range of
staining capability
o It is able to stain
cell nuclei and platelet granules
Most of the Romanowsky stains are prepared with _________ so that they act as a fixative as well as
the cellular stain.
Methyl alcohol (Methanol)
There are 4 different types of Romanowsky stains
commonly used in Hematology laboratory for staining the
blood cells:
o Wright’s Stain
o Giemsa Stain
o Leishman Stain
o Field’s Stain
Azure compounds with methylene blue and eosin,
aqueous stain
Best for inclusion bodies
Giemsa (1904)
Giemsa In combination with Jenner or May Grunwald
stains it constitutes
“Panoptic stain”
An_________ means it has no alcohol
accustain
Constitutes combination of a Romanowsky stain
with another stain; such that it improves staining
of cytoplasmic granules and other bodies:
▪ Jenner-Giemsa
▪ May Grunwald-Giemsa
Panoptic Staining
is a mixture of Methylene blue, and Eosin
dye, prepared in Alcohol medium and diluted with buffer or distilled water during staining procedure.
Leishman stain
The Leishman stain is one of the best stains for routine
blood stain to stain the Peripheral blood smear for the
examinations of blood film under the microscope and is
satisfactory for malaria and other blood parasites.
o ___________ gives better results in parasitic
studies.
Leishman stain, Giemsa stain
It stains the different components of blood in a
range of shades between red and blue.
• It is based on a methanolic mixture of “polychromed”
Methylene blue and eosin
Leishman stain
