Peripheral Blood Smear (PBS) Flashcards by Kyra Catedral

• Purpose:
• Specimen:

To examine blood cells (rbc, wbc, and platelets) under the microscope to diagnose a disease.

Whole blood

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can be used but not preferred; EDTA blood reveals better morphology

SODIUM CITRATE

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SODIUM CITRATE tubes are used when ther eis presence of

platelet satellitosis

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_________ 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)

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______\ 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

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occurs when platelet agglutinates are similar in size to WBCs and automated analyzers cannot distinguish the two.

Pseudoleukocytosis

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Preparation of Smear (3)

Wedge
Cover Glass
Automated

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Manual wedge technique

2 slides used

Film slide
Spreader slide

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• EASIEST TO MASTER
• Convenient and commonly used

MANUAL WEDGE TECHNIQUE

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MANUAL WEDGE TECHNIQUE

2 types

Push
Pull

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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

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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

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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

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Dimension if glass slide

3 x 1 inches

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Space before smear
Smear size
Space after smear

0.5 in

2/3 or 3/4 of the slide

0.25 in or 1cm

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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)

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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

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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

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Thick Smears ASS

Too high angle
Too large drop of blood
Too fast

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Thin Smears ASS

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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.

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eight-point star pattern

• The______ (Sysmex Corporation of America, Mundelein, IL) is an automated slide-making and
-staining system

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Sysmex SP-10

STAINING OF PERIPHERAL BLOOD FILMS
PURPOSE:

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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_____

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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

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

1. RBCs are uniformly and singly distributed 2. Few RBC are touching or overlapping 3. Normal biconcave appearance 4. 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 >> 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 >> 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

Peripheral Blood Smear (PBS) Flashcards

(49 cards)