1.1 Blood Smear Preparation Flashcards
It is required for the enumeration and checking of cellular elements
Peripheral blood film
T or F
The peripheral blood film must be prepared immediately as possible
True
The peripheral blood film mostly uses what techniques (using EDTA) and Wright-GIEMSA stain
Wedge technique
Two-slide or Wedge menthol is also known as
Push-type wedge preparation
It is the simplest and the most common method of smear preparation
Two-slide / wedge method
It uses 2 slides:
- one for the the smear
- one that serves as a spreader
Wedge method / two slide methif
In this method, Hugh-quality, beveled-edged microscope slides are recommended.
Wedge method/ two slide method
It this wedge method type, spreader slide is pushed into the drop of blood and pulled along the length of the slide to make the film
Pulled-type
Wedge method
The small drop of blood on the slide must be ____ and diameter and ____ from the end of the slide
2-3 cm in diameter
1 cm from the edge
Wedge mehtid
Using the thumb and forefinger of the right hand, hold the end of the spreader against the surface of the smear slide at ____________ angle
30 - 45 degree angle
What are the features of a properly made peripheral blood film (6)
- The film must cover 2/3 to 3/4 the length of the slide.
- The film is finger shaped. Slightly rounded at the feathery edge, not bullet shaped.
- The lateral edges of the film are visible.
- The film is smooth without irregularities, holes or streaks
- When the slide is held up to the light, the feathery edge/thin portion of the film has rainbow appearance.
- The whole drop of the blood is picked up and spread.
What are some causes of unacceptable peripheral blood film?
- hesitation
- chipped/rough edge
- pushed too quickly
drop of blood was too small - drop of blood was not spread in the spreader slide
- dirt, grease, or elevated lipids
- uneven pressure
- time delay
Many holes in the slide may be attributed to
Dirty slides or grease or elevated lipped
What are the causes of a thin smear
- Small drop of blood
- angle is too small
- px has low hematicrut
What are the causes of a thick smear
- big drop of blood
- angle is too high
- px has high hematocrit
If the px has high hematocrit levels
Decrease the angle
If the px has low hematocrit levels (px has polycythemia or is a newborn)
Increase the angke
Ridges / waves in the slide are caused by
- hesitation = uneven spread
- too much oressure
What causes chipped / rigged edge
- unreliable spreader slide
- spreader has stopped near the end of the slife
A good film includes a 2 portions
Thick portion and think portion
Moving the pusher slide forward too slowly accentuates ______________ by pushing larger cells, such as monocytes and granulocytes, to the very end and sides of the film
poor leukocyte distribution
Slow drying like in humid environment will result to the formation of ______
Artifactual cells
The slide may be labeled with a lead pencil on the ________ or __________
- Frosted edge
- thicker end of the blood film must be
Why are balloons/markers not used when labeling slides
They may be washed out during staning
How can the thickness of the film be adjusted?
- changing the angle
- change of speed
- smaller/larger drop of blood was
Increasing the angle of the spreader slide will
Increase the thickness of the film
increasing the speed with which the spreader slide is pushed will
Increase the thickness of the film
It is an older technique that is now used only rarely for PBS, but it is sometimes still used for making bone marrow aspirate smear
Ehrlich’s Two-Coverglass method / Two-cover slip
It is used for making bone marrow aspirate smears since it requires only a small amount of sample
Ehrlich’s Two-Coverglass method / Two-cover slip
For ehrlich two cover glass method, no. 1 or 1 ½ cover glasses _________ square are recommended.
22 mm
In the ehrlich’s two cover glass method, if done correctly, both coverslips will have quality smears that will appear similar to a ________ print.
thumb print
Making smears in coverslips (ehrlich’s two coverglass method) requires
Manual dexterity
One of the advantages of the Erlich’s method is that it requires _________ amount of bone marrow
Minimal amount
It produces an excellent leukocyte distribution which lends itself to more accurate determination of differential counts
Ehrlich’s Two-Coverglass Method
One of the disadvantages of the Ehrlich’s Two-Coverglass Method is that coverslips are
Fragile and prone to breakage when storing it
In the Ehrlich’s Two-Coverglass Method, the two coverslips must form a _______ figure to begin spreading the blood
16-slides figure
Blood films that combined the advantages of easy handling of the wedge slide and uniform distribution of cells of the cover glass preparation, may be made with special types of centrifuges known as
Spinner
This produces a uniform blood film, in which all cells are separated (a monolayer) and randomly distributed
Spinner slide
Smears prepared using this method have white blood cells that can be easily identified at any spot in the film
Spinner method
On a wedge smear, the disproportionate of monocytes occur at the _______
Tip of a the feathered edge
On a wedge smear, the neutrophils are seen just in from the
Feathered edge
On a wedge smear, both neutrophils and monocytes may occurs at the
Lateral edges of the film
It is an automated slide making and staining system
Sysmex SP-1000i
One of the features of the Sysmex SP-1000i is that it adjusts the size of the drop of blood used and the angle and speed of the spreader slide in making a wedge preparation
Dependent in hematocrit levels
In Sysmex SP-1000i, a film is produced for every
30 seconds
T or F
In the Sysmex SP-1000i, the slide comes with a printed label: patient name, number, and date
True
This automated smear preparation machine prepares slides automatically based on orders received from the LIS
DxH Slidemaker Stainer II Cellular Analysis system
This automated smear preparation machine create and stain smears from capped whole blood tubes, open-top tubes or pediatric whole blood tubes
DxH slidemaker Stainer II Cellular Analysis System
In the DxH Slidemaker Stainer II Cellular Analysis System, how many slides are made from a single 90 uL sample and from a single sample presentation
90 uL = 4 slides
1 sample = up to 12 slides
It can create multiple stain smear slides, according to any parameter results, such as a low WBC or Blast suspect flag
DxH Slidemaker Stainer II Cellular Analysis System
What is the anticoagulant of choice for hematology cell counts and cell morphology
EDTA (ethylenediaminetetraacetic acid)
What Romanowsky stains are used for staining of peripheral blood film
Pure weight stain or Wright-GIEMSA Stain
It is a methylalcholic solution of eosin & a complex mixture of thyocin (methylene blue, assure blue, etc.)
Romanowsky stain
Stain that is most commonly used for staining peripheral blood and bone marrow smears
Romanowsky stain
Romanowsky stains are polychrome stains that contain
Eosinophils and methylene blue
What are the two anticoagulant not allowed to be used when using wright’s stain
Heparin - causes blue bg
Oxalate & Fluoride - distort morphology
Heparin is not allowed when staining using Wright’s staining because it causes
Blue background
Oxalate & Fluoride are not allowed when staining using Wright’s staining because it causes
Distortion of morphology
Slides are manually smeared by dipping into the reagents found in coplin jar
Staining jar or dip method
Enumerate the steps of the Staining jar or dip method: Wright staining
- Methanol - 30 seconds
- Eosin - 6 seconds
- Methylene blue - 4 seconds
Buffer solution/aged distilled water - 45 seconds
In the dip method, the fixative used to fix the cells to the glass slide is (1st step)
Methanol - 30 seconds Buffer solution
In the dip method, the seconds step involves free eosin which are acidic and stain basic cellular components such as
Hemoglobin or eosinophilic granules (appear red)
In the dip method, the third step involves free methylene blue which are basic and stain acidic cellular components such as
RNA (appear blue)
In the dip method, the 4th step requires ______________ or aged distilled water (distilled water placed in a glass bottle for at least 24 hours; pH 6.4 to 6.8)
- 0.05 M sodium phosphate (pH 6.4)
- aged distilled water (24 hrs) (pH 6.4 - 6.8)
In an optimally stained peripheral blood film, the RBCs should appear
Pink to sslmon
In an optimally stained peripheral blood film, the nuclei should appear
Dark blue or purple
In an optimally stained peripheral blood film, the cytoplasmic granules of neutrophils appear
Lavender to lilac
In an optimally stained peripheral blood film, the cytoplasmic granules of basophils appears
Dark blue to black
In an optimally stained peripheral blood film, the cytoplasmic granules of eosinophils appear
Red to orange
In an optimally stained peripheral blood film, the area between the cells should be
Colorless, clean, free of precipitated stain
The best staining results are obtained from freshly made slides that have been prepared within _________ hours of blood collection.
2 to 3 hrs
What could be the cause of this blood film appearance?
RBCs appear gray/blue/green (should be pink or salmon)
- stain or buffer is too alkaline
- inadequate rinsing
- prolonged staining
- heparinized blood sample
What could be the cause of this blood film appearance?
The nuclear chromatin appears deep blue to black (should be dark blue to purple)
- stain or buffer is too alkaline
- inadequate rinsing
- prolonged staining
- heparinized blood sample
What could be the cause of this blood film appearance?
Eosinophils granules are blue/gray not red - orange
- stain or buffer is too alkaline
- inadequate rinsing
- prolonged staining
- heparinized blood sample
What can you do to troubleshoot too alkaline slides, inadequate rinsing, prolonged staining, or heparinized blood sample
- staining for a shorter time or using less stain
and more diluent may correct the problem - buffer may be too alkaline, and a new one with a lower pH should be prepared
- replace the reagents with new ones in its optimal state
What could be the cause of this blood film appearance?
RBCs appear too pale or bright
- stain or buffer too acidic (most common)
- underbuffering (time too short)
- over-rinsing
What could be the cause of this blood film appearance?
WBCs appear too dark
- Stain or buffer too alkaline (most common)
- Inadequate rinsing
- Prolonged staining
- Heparinized blood sample
What could be the cause of this blood film appearance?
Neutrophils are deeply overstated, large, and prominent
- stain or buffer is too alkaline
- inadequate rinsing
- prolonged staining
- heparinized blood sample
What could be the cause of this blood film appearance?
Nuclear chromatin appears pale blue
- stain or buffer too acidic (most common)
- Underbuffering (time too short)
- Over-rinsing
- exposure to acid fumes
What could be the cause of this blood film appearance?
WBCs are barely visible
- stain or buffer too acidic (most common)
- Underbuffering (time too short)
- Over-rinsing
- exposure to acid fumes
What could be the cause of this blood film appearance?
Eosinophils appear to have sparkling brilliant red granules
- stain or buffer too acidic (most common)
- Underbuffering (time too short)
- Over-rinsing
- exposure to acid fumes
What can you do to troubleshoot too acidic (most common), underbuffering, over-rinsing, exposure to acid fuse
- Prevent the exposure of stains or buffer to acid fumes as this may result to over acidic reagents
- Low pH of the buffer/methyl alcohol may lead to the development of formic acid as a result of oxidation on standing
- Replace the reagents with new ones in its optimal state
Inadequately stained red cells, nuclei, or eosinophilic granules may be due to
- under staining
- excessive washing
Presence of precipitates on the film due to
Unclean slides
What are other causes of problems for PBS
- failure to hold the slide horizontally during initial washing
- inadequate filtration of the stain
- permitting dust to settle con the slide/smear
Giemsa Stainer is an excellent stain for _____ parasite
Malarial parasite
What are other romanowsky-type stain other than wrights stain
- giemsa stain
- leishman’s syain (flagella)
- jenner’s
- may-grünwald
- macneal’s
