PBS Flashcards
When should blood smears be made from EDTA blood?
Within 3 hours after collection
Advantages of EDTA blood smear
Multiple smears, delayed preparation, prevents platelet clumping
Disadvantages of EDTA blood smear
Platelet satellitism, EDTA-induced platelet clumping
What conditions can EDTA cause in PBS?
Pseudothrombocytopenia, pseudoleukocytosis
Correction for EDTA effects in PLT
Recollect using 3.2% Na Citrate, multiply platelet count by 1.1
Best specimen for evaluating cell morphology?
Anticoagulant-free blood
Advantages of anticoagulant-free blood
Prepared at patient’s side, prevents some artifacts
Disadvantages of anticoagulant-free blood
Platelet clumping (caused by glass), limited films can be made
Most frequently used blood film preparation method?
Two-glass slide method (Manual Wedge Technique)
What angle should be between the pusher and film slide?
30 - 45 degrees
Effect of too high angle of spreader?
Thicker smear
Effect of too low angle of spreader?
Thinner smear
Ideal size of blood drop for smear?
2-3mm
Effect of too large blood drop?
Thicker smear
Effect of too small blood drop?
Thinner smear
Distance from label when preparing smear?
1 cm away
Effect of too fast spreader speed?
Thicker smear
Effect of too slow spreader speed?
Thinner smear, poor WBC distribution
Effect of high hematocrit (Hct) correction in PBS?
Lower the angle (as low as 25 degrees)
Effect of low hematocrit (Hct) correction in PBS?
Raise the angle
How should the scanning method be done under microscope?
Longitudinal (tail to head) or battlement (back and forth serpentine)
What is the ideal transition of a blood smear from thick to thin area?
Gradual transition
How much of the film slide should the smear cover?
2/3 to 3/4 of the length of the film slide
What shape should the smear be?
Finger-shaped
What should be visible on the smear?
Lateral edges
What should the smear not have?
Irregularities, holes, or streaks
What appearance should the feather edge have?
Rainbow appearance
What problem occurs with Wedge method?
WBCs are unevenly distributed across the slide
Where are segmented neutrophils, monocytes, and eosinophils more visible?
In the feather edge and side edges
Where are small lymphocytes more visible?
At the center of the film
What is the purpose of blood smear staining?
Evaluation of cellular morphology
What is the fixative used in blood smear staining?
Methanol
What buffer solution is used for blood smear staining?
0.05 M NaPO4 at pH 6.4
What is the pH range for blood smear staining?
6.4 to 6.8
What is the commonly used stain for blood smears?
Wright/Wright Giemsa stain
What is a Romanowsky-type stain?
A stain containing methylene blue and eosin B or eosin Y
What is the function of methylene blue in staining?
It is a basic stain that colors the nucleus and some cytoplasmic structures blue or purple
What structures are described as basophilic?
DNA or RNA
What is the function of eosin in staining?
It is an acidic stain that colors cytoplasmic structures orange-red
What structures are described as acidophilic?
Proteins with amino groups
What are some examples of Romanowsky-based stains?
Wright stain, Giemsa stain, May-Grunwald stain
What automated method for PBS does not require a power source?
CellaVision HemaPrep®
What type of blood is used in centrifugal (spinner) type preparation?
0.2 mL of well-mixed anticoagulated blood
What is a key advantage of the centrifugal (spinner) type method?
Even distribution of blood cells; consistent; fewer smudge cells
Other types of automated methods for PBS preparation
Coulter LH, Sysmex SP-10
Macroscopic characteristic of a well-stained blood smear
Pink to Purple
RBC color in a well-stained blood smear
Orange to Salmon-pink
WBC nuclei color in a well-stained blood smear
Purple-blue
Neutrophil cytoplasm color in a well-stained blood smear
Pink to Tan (granules violet to lilac)
Eosinophil granules color in a well-stained blood smear
Bright orange
RBCs appearing gray or blue in smear
Staining buffer too basic, inadequate rinsing, heparinized blood used
WBCs appearing too dark in smear
Staining buffer too basic, inadequate rinsing, heparinized blood used
Eosinophil granules appearing gray in smear
Staining buffer too basic, inadequate rinsing, heparinized blood used
RBCs appearing too pale or red in smear
Stain buffer too acidic, under buffering, over rinsing
WBCs barely visible in smear
Stain buffer too acidic, under buffering, over rinsing
Blood film appearing bluer than normal
Increased blood proteins (e.g., plasma cell myeloma)
Grainy appearance on blood film
RBC agglutination (e.g., cold hemagglutinin disease)
Holes in blood film
Increased lipid levels
Blue specks at feather edge of blood film
Markedly increased WBC and platelet counts
10x Objective Use in microscopic examination of PBS
Assess overall film quality, color, and cell distribution; locate rare abnormal WBCs; detect “snowplow” effect, fibrin strands, rouleaux formation, RBC agglutination, parasites; assess examination area
Snowplow effect indication
More than four times the number of WBCs per field at the lateral edges or feather edge compared with the monolayer area
Fibrin strands detection
Film should be rejected if present
40x High-Dry or 50x Oil Immersion Objective Use
Examine areas where red cells have central pallor, are near each other but not overlapping; cells are appropriately stained; avoid feathered edge and thick part
Feathered edge appearance under higher magnification
Red cells appear macrocytic, flattened, and lack central pallor; WBCs distorted
Thick part appearance under higher magnification
Red cells appear microcytic and may form rouleaux
Method for estimating total WBC count using 40x high-dry objective
Count leukocytes in 10 fields, find the average number of WBCs per field, then multiply by 2,000 to get estimated WBC count per μL
Method for estimating total WBC count using 50x oil immersion objective
Count leukocytes in 10 fields, find the average number of WBCs per field, then multiply by 3,000 to get estimated WBC count per μL
100x Oil Immersion Objective Use
Examine nuclear details of white blood cells; tabulate WBC differential; estimate platelet count
RBC count per 100x OIF
200 to 250 RBCs per 100x OIF in normal smear
Method for estimating platelet count using 100x oil immersion objective
Scan ten oil immersion fields, average platelets per field, multiply by 20,000 for estimated plt. ct. per μL
Platelet estimate formula in significant anemia or erythrocytosis
Average number of platelets per field x total RBC count / 200
Storage duration for blood smear slides
At least 7 days before proper disposal