Lecture 1 Flashcards by akanksha patel

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)

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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

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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

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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

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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

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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

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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

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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 4C for up to 12 hours minimizes changes

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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

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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

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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

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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

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unacceptable smears

rough or chipped edge
not along the whole slide
blood drop too small/large
streaks - uneven pressure

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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.

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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

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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

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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.

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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)

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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