Peripheral Blood Smear (PBS) Flashcards
• Purpose:
• Specimen:
To examine blood cells (rbc, wbc, and platelets) under the microscope to diagnose a disease.
Whole blood
can be used but not preferred; EDTA blood reveals better morphology
SODIUM CITRATE
SODIUM CITRATE tubes are used when ther eis presence of
platelet satellitosis
_________ when anticoagulated with EDTA.
• The platelets surround or adhere to neutrophils, which potentially causes_______ when counting is done by automated methods
• In addition, spuriously low platelet counts and _______can result from EDTA- induced platelet clumping.
platelet satellitosis
pseudothrombocytopenia
falsely increased WBC counts (pseudoleukocytosis)
______\ EDTAis often preferred to the powdered form because it mixes more easily with blood.
High-quality blood films can be made from the blood in the EDTA tube, provided that they are made within 2 to 3 hours of drawing the specimen!
Liquid tripotassium EDTA
K3
occurs when platelet agglutinates are similar in size to WBCs and automated analyzers cannot distinguish the two.
Pseudoleukocytosis
Preparation of Smear (3)
Wedge
Cover Glass
Automated
Manual wedge technique
2 slides used
Film slide
Spreader slide
• EASIEST TO MASTER
• Convenient and commonly used
MANUAL WEDGE TECHNIQUE
MANUAL WEDGE TECHNIQUE
2 types
Push
Pull
Tips to make a Good blood film
SPA
• The_______: too large a drop creates a long or thick film, and too small a drop often makes a short or thin blood film.
• Maintaining an ________on the slide is essential.
• It is also crucial to ______all the way to the end of the film
• If two or three films are made, the best one is chosen for staining, and the others are disposed of properly.
Some laboratories require two good films and save one unstained in case another slide is required.
SIZE OF THE DROP of blood is important
even, gentle pressure
keep the same angle
Manual wedge technique
PROCEDURE
1. A drop of blood (_____in diameter) is placed at one end of the smear slide.
a.______ (used in malarial smear because it needs thick and thin smear)
b._____ (Much better)
- The spreader slide is placed at about a ______ angle against the smear slide (Figure A)
- The______ is drawn back into the drop of blood, and the blood is allowed to spread across the width of the slide (Figure B)
- The spreader is then quickly and smoothly pushed forward at the end of the smear slide to create a wedge film (Figure C).
5.______
2-3 mm
Grounded Slide
Frosted slide
30-45°
spreader
Margin-free
Manual wedge technique
PROCEDURE
1. A drop of blood (_____in diameter) is placed at one end of the smear slide.
a.______ (used in malarial smear because it needs thick and thin smear)
b._____ (Much better)
- The spreader slide is placed at about a ______ angle against the smear slide (Figure A)
- The______ is drawn back into the drop of blood, and the blood is allowed to spread across the width of the slide (Figure B)
- The spreader is then quickly and smoothly pushed forward at the end of the smear slide to create a wedge film (Figure C).
5.______
2-3 mm
Grounded Slide
Frosted slide
30-45°
spreader
Margin-free
Dimension if glass slide
3 x 1 inches
Space before smear
Smear size
Space after smear
0.5 in
2/3 or 3/4 of the slide
0.25 in or 1cm
Features of a well-made PBS
- The film is_______ the length of the slide.
- The film is_____ shaped, very _____at the feather edge. NOT____ shaped; this provides the widest area for examination.
- The lateral edges of the film are visible.
- The film is smooth without (3).
- When the slide is held up to the light, the thin portion (feather edge) of the film has a “____” appearance.
- The_____ is picked up and spread.
two thirds to three fourths
finger; slightly rounded ; bullet
irregularities, holes, or streaks
rainbow
whole drop of blood (bawala ng naay tutoy)
Unacceptable Peripheral Blood Smear
A. Chipped or rough edge on spreader slide
B. Hesitation in forward motion of spreader slide
C. Spreader slide pushed too quickly
D. Drop of blood too small
E. Drop of blood not allowed to spread across the width of the slide
F. Dirt or grease on the slide; may also be due to elevated lipids in the blood specimen
G. Uneven pressure on the spreader slide
H. Time delay; drop of blood began to dry
FACTORS THAT DETERMINE THE THICKNESS OF SMEAR (PASS)
Pressure of spreader on slide
Angle between slide and spreader
Size of blood drop
Speed of spreader
Thick Smears ASS
Too high angle
Too large drop of blood
Too fast
Thin Smears ASS
Too low angle
Too small drop of blood
Too slow
COVER GLASS METHOD
Procedure
1. Touch a cover glass to the top of a small drop of blood without touching the skin
2. Place the blood side of the cover glass face down, crosswise, on another cover glass so that the corners form an_____
3. If the drop of blood is not too large and the cover glasses are perfectly clean, the blood will spread out evenly and quickly in a thin layer between the two surfaces.
4. As the blood spreads and just before it stops, quickly but firmly pull the cover glasses apart on a plane parallel to their surfaces
5. The blood will usually spread more evenly on one of the cover glasses than the other
6. Place the cover glasses film side up on clean paper and allow them to air dry or you may insert them back to back in slits made in a cardboard box for drying.
eight-point star pattern
• The______ (Sysmex Corporation of America, Mundelein, IL) is an automated slide-making and
-staining system
Sysmex SP-10
STAINING OF PERIPHERAL BLOOD FILMS
PURPOSE:
to make the cells more visible and to allow their morphology to be evaluated
STAINING OF PERIPHERAL BLOOD FILMS
PRINCIPLE:
• ______utilized in staining blood smears fall into two main categories: (2)??
• When stained by basic dyes, NUCLEI and specific other structures in the blood are termed_____
• Structures that exclusively take up acid dyes are called_____
• Some structures are stained by a combination of both types are are referred to as_____
Aniline dyes
BASIC DYES (Methylene blue) and ACID DYES (Eosin)
BASOPHILIC
ACIDOPHILIC or EOSINOPHILIC
NEUTROPHILIC
Romanowsky Stain
• Polychromatic Stain
• Giemsa Stain
• Wright’s stain
• Quick stain
• Polychromatic Stain
• Methylene blue
• Eosin
Classifying LYMPHOMAS in the KIEL CLASSIFICATION
Giemsa Stain
Plasmodium classification
Giemsa Stain
Used also in histology
Giemsa Stain
Demonstration of parasites in MALARIA
Giemsa Stain
Giemsa Stain
Demonstration of parasites in MALARIA
Used also in histology
Plasmodium
Classifying LYMPHOMAS in the KIEL CLASSIFICATION
Wright Stain
• Differentiation of blood cell types
• Peripheral Blood Smear
• Manual & Automated
• Manual & Automated
Wright stain
Stain
• Peripheral Blood Smear
Wright stain
• Differentiation of blood cell types
Wright stain
Procedure for wright stain
1. Blood smears must be stained within a few hours of preparation or fixed with methanol if they must be kept without staining.
2. Fixation and staining may be done by immersing the slides in reagent-filled coplin jars or by covering the horizontally positioned slides with the reagents.
3. FIXATION with ABSOLUTE METHANOL for 1-2 minutes.
4. Expose the slide to the stain solution for 2 minutes.
5. Without removing the stain from the horizontal slide, an equal amount of buffer is carefully added and is mixed by blowing gently.
6. The stain is flushed from the horizontal slide with water. NOTE: Washing for longer than 30 seconds reduces the blue staining.
7. Clean the back of the slide with gauze.
8. Allow the slide to air dry in a tilted position.
9. Cover slips may be mounted to protect the film.
_______, as the name implies, are fast and easy. The whole process takes about 1 minute. The stain is purchased in a bottle as a modified Wright or Wright-Giemsa stain. The required quantity can be filtered into a Coplin jar or a staining dish, depending on the quantity of slides to be stained.
Quick stains
PERIPHERAL FILM EXAMINATION
Macroscopic
• Bluer film
• Grainy appearance
• Holes all over the film
Peripheral Film Examination
• Microscopic
- overall film quality, color, and distribution of cells can be assessed
• 10x objective
Peripheral Film Examination
• Microscopic
- easy to select the correct area of the film in which to begin the differential count and to evaluate cellular morphology
• 40x objective
Peripheral Film Examination
• Microscopic
- WBC differential and morphology examinations described for the 100x oil immersion objective also can be accomplished by experienced morphologists
• 50x objective
Peripheral Film Examination
• Microscopic
- WBC differential count generally is performed
• 100x objective
Optimal Assessment Area
- RBCs are uniformly and singly distributed
- Few RBC are touching or overlapping
- Normal biconcave appearance
- 200 to 250 RBC per 100x OlO
METHODS FOR DIFFERENTIAL COUNT
BATTLE-MENT METHOD
LONGITUDINAL STRIP METHOD
STEP TECHNIQUE
TOO ACIDIC SMEARS
STAINING ISSUES
Insufficient staining time
Prolonged buffering or washing
Buffer or water may be due to exposure of stain, buffer to acid fumes or it may be due to old stain in which the methyl alcohol has slowly
Prolonging staining time
Shorten buffering time
Check the pH of stain and buffer and correct with alkali if necessary
TOO ACIDIC SMEARS
STAINING ISSUES
Insufficient staining time
Prolonged buffering or washing
Buffer or water may be due to exposure of stain, buffer to acid fumes or it may be due to old stain in which the methyl alcohol has slowly
TOO ACIDIC SMEARS
STAINING ISSUES
Insufficient staining time
Prolonged buffering or washing
Buffer or water may be due to exposure of stain, buffer to acid fumes or it may be due to old stain in which the methyl alcohol has slowly
TOO ALKALINE SMEARS
STAINING ISSUES
REMEDIES
Thick blood smears»_space;
Prolonged staining time
Insufficient washing
Too alkaline pH of stain, buffer, or water
New stain solution which has not stood for 2-3 weeks may be too basic
Shorten staining time
Prolong buffering time
Check the pH of stain, buffer, and water
Check the incubation time of the stain
TOO ALKALINE SMEARS
STAINING ISSUES
Thick blood smears»_space;
Prolonged staining time
Insufficient washing
Too alkaline pH of stain, buffer, or water
New stain solution which has not stood for 2-3 weeks may be too basic
