Lecture 1 Flashcards
What’s in Blood
Plasma – liquid portion of blood which transports and nourishes the cells - proteins, fibrinogen
Cellular components – consist of the RBCs -Erythrocytes , WBCs -Leukocyte, and PLTs- Thrombocytes
the blood at the bottom - packed red cells -hematocrit
able to provide the physician with CBC (rbc, wbc and plts)
Hematology Workflow
Physician orders a CBC (panel of tests)
Sample is collected
Sample is run on automated cell counter
Automated results are reported or a PBF is required
Blood film is made on glass slide
Blood film is stained
Blood film is coverslipped
Microscopic evaluation of PBF (peripheral blood film) is preformed
Acceptable Specimens for Peripheral Blood Films
Venipuncture specimens – whole blood taken from veins (preferred sample)
Capillary blood from finger-stick or heel-stick are acceptable – smears made at beside before blood clots
2 identifiers for identifying blood
Sample must be anticoagulated
Stops blood from clotting
If sample clotted, cells are bound in the clot and cannot be assessed
EDTA (lavender top tube) is the anticoagulant of choice for routine Hematology testing
Must be immediately, gently and thoroughly mixed when tube is filled
EDTA Whole Blood Sample
EDTA = Ethylenediaminetetraacetic acid
Chelates calcium
Calcium is necessary in the coagulation cascade- its removal inhibits a series of events which cause clotting
Preserves cellular components and morphology (least amount of cell distortion)
Collection tubes with EDTA should be mixed by 8-10 end-to-end inversions immediately following venipuncture collection
Microcollection tubes with EDTA should be mixed 10 times end to end inversions
Blood Stability, Storage & Retention
EDTA specimens should be analyzed within five hours of collection at room temperature.
Microcollection EDTA specimens should be analyzed within four hours of collection at room temperature
smears should be done in 4 hours - reduce cell deterioration and artifacts in morph
put in fridge okay in room temp for 24 hours and time retained is facility based
Unacceptable Specimens for CBC & PBFs
Partially or Under-Filled Tubes
Blood : Anticoagulant ratio is important
Too-little blood = too much EDTA
Tube must be filled ± 10% of the stated draw volume
Excess EDTA leads to:
Erroneously low blood cell counts and Hematocrits
Staining alteration
Morphologic changes to RBCs and WBCs on blood film
RBC and WBC shrinkage
Crenated RBCs
WBC membrane damage
Overexposure to EDTA - left out too long
Cells in EDTA > 5 hours at RT show artifacts:
Crenated RBCs (pie crust membrane), Spherocytes
MCV - Can be increased due to RBC swelling
HCT - Can be increased due to increases in MCV
Necrobiotic/necrotic (dying) WBCs (decreasing counts)
Progressively vacuolated WBCs (especially Neutrophils) clear stained area in cytoplasm
Storage at 4C for up to 12 hours minimizes changes
Over-Filled Tubes
Would only occur if filling EDTA tube from a syringe
Prevents proper mixing of sample = Insufficient EDTA for blood volume
May lead to platelet clumping and clotting
Clotted sample – Reject specimen
Tube must be filled ± 10% of the stated draw volume
Incorrect Phlebotomy Technique
Slow/difficult venipuncture draw, improperly mixed tubes or improper handling means blood may not get exposed to EDTA in time
Small clots (fibrin strands or microclots) or clumping platelets may affect patient results
You can’t tell this from the tube – you must make a PBF
Automated platelet count may be unexpectedly (falsely) decreased
what do we look for Body of the smear
not too thin or thick – this is where we examine the cells
Feathered Edge’ or tail at the thinnest edge of the smear
when a smear is well made
2/3-3/4 of the slide
round or feathered edge that has a rainbow appearance
thinner on the side
smooth appearance
the whole drop of blood is picked up no dry spots
unacceptable smears
rough or chipped edge
not along the whole slide
blood drop too small/large
streaks - uneven pressure
The drop of blood is too large (long, thick smear), too small (short smear) or too much left behind (dried circle at end of smear
adjust size of drop of blood accordingly.
The spreader is pushed with a jerky motion and/or lifted off the slide (banding on smear)
hold spreader firmly against slide and use a smooth motion with even pressure
The spreader is not pushed rapidly enough
smear may be too long and larger WBC such as, monocytes and granulocytes, are pushed to the sides/end of smear) – adjust speed accordingly
The blood in the capillary tube has dried up and does not flow – need to ‘tap’ to get blood out
DISCARD capillary tube and use a new one.
Thickness & length of the smear is influenced by the spreader angle
Smaller angle – longer, thinner smear
if too short- adjust angle of spreader down or use larger drop (gives longer, thicker smear)
Larger angle – shorter, thicker smear
if too long – adjust angle of spreader up or use smaller drop (gives shorter, thin smear)
Hemoglobin, Hematocrit or RBC count very low - technique issue
smear is very long and thin. Adjust the angle up to shorten smear.
Hemoglobin, Hematocrit or RBC count is high - technique issue
smear is short and thick. Adjust the angle down to lengthen the smear
Drying Blood Films
Reduced air humidity
Don’t blow on slides or wave slides around
Lay slides flat or stand upright
Slides must be completely dry before staining
If not, head of smear will wash off during staining process or
Artifacts may appear on smear
Water or Drying artifact
occurs if slides dry too slowly or too much moisture/humidity is present
RBCs look ‘moth-eaten’ (holes) or crenated (shrunken)
holes in film ifsample is lipemic
Central pallor of some RBCs are too distinct – ‘punched out’ artifact
Refractive (shiny) rings on RBCs
Extremely anemic samples – increased Plasma:RBC ratio
Excess moisture – hard to avoid drying artifact
Staining Peripheral Blood Films
stains what
Specifically stains cytoplasm, nucleus and other cellular components
Demonstrates the shape and hemoglobin content in RBCs
Differentiates the various types of WBCs
Visualize PLTs
Romanowsky stains include:
used for peripheral blood and bone marrow smears
Wright - most common
Wright-Giemsa - most common
Jenner
Leishman
Romanowsky stains are
“polychromatic”
“Poly” = many or much
“Chrom” = colour
Range of colours between red and blue
Alcoholic stain
Pure Wright stain or Wright-Giemsa
They are considered ‘polychrome’ stains because they contain both Eosin (Y or B) & Methylene Blue
Giemsa stain also contains Methylene Blue Azure
Best for staining early RBCs with excess RNA
Wright’s Stain
Methanol fixes the cells to the slide (added prior to staining especially if staining delayed or in the stain)
add po4 buffer pH of 6.4 – 6.8 to stain mix on the frosted end until a green metallic sheen forms
Oxidized methylene blue and eosin form a thiazine-eosinate complex which stains the neutral components
tissue that uses acidic(-) dye binds to basic (+) charged tissue - acidophilic
tissue that uses basic (+) dye binds to acidic (-) charged tissue - basophilic
Principle of Wright’s Stain
Free methylene blue is basic and stains acidic cellular components from blue-grey to dark blue colour:
i.e., Nucleic Acids: RNA & DNA, and Basophil granules
Free eosin is acidic and stains basic cellular components from light to deep pink to red or orange colour:
i.e., Hemoglobin and Eosinophilic granules
Neutrophils have cytoplasmic granules with neutral pH and pick up some staining characteristics from both stains (thiazine-eosinate complex)
Neutrophil granules appear pink/violet (lavender to lilac)
Mechanism of StainingIonic bonding and Buffer pH
Add acid (H+) to solution = more +ve charges, more attachment sites for acid dye
Stronger eosin staining – RBC and eosin too red
Weaker methylene blue staining – WBC/nuclei too pale
Add base (OH-) to solution = more -ve charges, more attachment sites for basic dye
Stronger methylene blue staining – nuclei/wbc very dark
Weaker eosin staining – RBC & Eosinophils look grey
Buffer pH must be between 6.4 to 6.8
Pink:
Neutrophil cytoplasm
RBCs (HGB- pink to salmon)
Eosinophil granules (can be red to orange as well)
Pink-Violet:
Neutrophilic granules
Platelet granules
Purple:
Nuclei (dark)
Purple or Red:
Azurophilic granules
Dark Purple-Black:
Basophilic granules
Blue:
Nuclei (dark)
Lymphocyte cytoplasm
Grey-Blue:
Monocyte cytoplasm
Features of a Optimally-Stained PBF
Macroscopically -
Smear should appear pink to purple
Microscopically –
RBCs are pink to salmon
Nuclei are dark blue to purple
Neutrophil cytoplasmic granules are lavender to lilac
Eosinophil cytoplasmic granules are red to orange
Basophil cytoplasmic granules are dark purple to black
Area between cells should be colourless, clean, and free of stain precipitates
Stain precipitates on slide
problem
Filtering of stain required
Insufficient rinsing (see SOP)
Allowing stain to dry on slides during staining process
problem
RBCs appear grey
Eosinophil granules are grey, not orange
WBC nuclei are too dark
Stain or buffer too alkaline (most common)
Inadequate rinsing
Prolonged staining
Heparinized blood sample
problem
RBCs too pale or red colour
WBC nuclei barely visible
Stain or buffer too acidic (most common)
Under-buffering (too short time)
Over-rinsing
Stain old
problem
Stain deposit
Stain needs to be filtered
Poor rinsing of slide
Stain dried on slide
Inadequate staining – WBC nuclei very pale
problem
Stain or buffer too acidic
Insufficient time in buffer (under-buffer)
Over-rinsing
Stain is too old
Used when rapid Turn-Around-Time (TAT) required
Commercially prepared, all-in-one Wright or Wright-Giemsa stain
Quality is often a concern, but timing can be slightly adjusted to attain quality colour
Coverslipping Slides
Good-quality glass cover slips are used to cover stained PBFs
Held in place by ‘mounting media’ –
Synthetic resins, such as, Permount
Protect smear while stored
Minimize biological hazard
Microscopes are designed to focus on slides with coverslips – slide & coverslip have same RI
Objectives show maximum coverslip thickness (in mm)
Essential to set up microscope for optimal slide viewing (Kohler Illumination)
If acceptable, label slide as follows:
Label using pencil (not pen or marker)
Label with frosted side to the top
Include:
Patient’s name (Last, First)
ID#
Date (YYYY-MM-DD) & Time (24hr xx:xx)
Your initials + MB#
Allow the film to air dry (lay flat) on bench
