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Hematology > Lecture 1 > Flashcards

Lecture 1 Flashcards

1
Q

What’s in Blood

A

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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2
Q

Hematology Workflow

A

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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3
Q

Acceptable Specimens for Peripheral Blood Films

A

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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4
Q

EDTA Whole Blood Sample

A

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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5
Q

Blood Stability, Storage & Retention

A

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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6
Q

Unacceptable Specimens for CBC & PBFs

A

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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7
Q

Excess EDTA leads to:

A

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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8
Q

Overexposure to EDTA - left out too long

A

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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9
Q

Over-Filled Tubes

A

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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10
Q

Incorrect Phlebotomy Technique

A

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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11
Q

what do we look for Body of the smear

A

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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12
Q

when a smear is well made

A

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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13
Q

unacceptable smears

A

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

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14
Q

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

A

adjust size of drop of blood accordingly.

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15
Q

The spreader is pushed with a jerky motion and/or lifted off the slide (banding on smear)

A

hold spreader firmly against slide and use a smooth motion with even pressure

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16
Q

The spreader is not pushed rapidly enough

A

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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17
Q

The blood in the capillary tube has dried up and does not flow – need to ‘tap’ to get blood out

A

DISCARD capillary tube and use a new one.

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18
Q

Thickness & length of the smear is influenced by the spreader angle

A

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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19
Q

Hemoglobin, Hematocrit or RBC count very low - technique issue

A

smear is very long and thin. Adjust the angle up to shorten smear.

20
Q

Hemoglobin, Hematocrit or RBC count is high - technique issue

A

smear is short and thick. Adjust the angle down to lengthen the smear

21
Q

Drying Blood Films

A

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

22
Q

Water or Drying artifact

A

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

23
Q

Staining Peripheral Blood Films
stains what

A

Specifically stains cytoplasm, nucleus and other cellular components

Demonstrates the shape and hemoglobin content in RBCs
Differentiates the various types of WBCs
Visualize PLTs

24
Q

Romanowsky stains include:

A

used for peripheral blood and bone marrow smears
Wright - most common
Wright-Giemsa - most common
Jenner
Leishman

25
Q

Romanowsky stains are

A

“polychromatic”
“Poly” = many or much
“Chrom” = colour

Range of colours between red and blue

Alcoholic stain

26
Q

Pure Wright stain or Wright-Giemsa

A

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

27
Q

Wright’s Stain

A

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

28
Q

Principle of Wright’s Stain

A

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)

29
Q

Mechanism of StainingIonic bonding and Buffer pH

A

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

30
Q

Pink:

A

Neutrophil cytoplasm
RBCs (HGB- pink to salmon)
Eosinophil granules (can be red to orange as well)

31
Q

Pink-Violet:

A

Neutrophilic granules
Platelet granules

32
Q

Purple:

A

Nuclei (dark)

33
Q

Purple or Red:

A

Azurophilic granules

34
Q

Dark Purple-Black:

A

Basophilic granules

35
Q

Blue:

A

Nuclei (dark)
Lymphocyte cytoplasm

36
Q

Grey-Blue:

A

Monocyte cytoplasm

37
Q

Features of a Optimally-Stained PBF

A

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

38
Q

Stain precipitates on slide
problem

A

Filtering of stain required
Insufficient rinsing (see SOP)
Allowing stain to dry on slides during staining process

39
Q

problem
RBCs appear grey
Eosinophil granules are grey, not orange

WBC nuclei are too dark

A

Stain or buffer too alkaline (most common)
Inadequate rinsing
Prolonged staining
Heparinized blood sample

40
Q

problem
RBCs too pale or red colour

WBC nuclei barely visible

A

Stain or buffer too acidic (most common)
Under-buffering (too short time)
Over-rinsing
Stain old

41
Q

problem
Stain deposit

A

Stain needs to be filtered
Poor rinsing of slide
Stain dried on slide

42
Q

Inadequate staining – WBC nuclei very pale

problem

A

Stain or buffer too acidic
Insufficient time in buffer (under-buffer)
Over-rinsing
Stain is too old

43
Q

Used when rapid Turn-Around-Time (TAT) required

A

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

44
Q

Coverslipping Slides

A

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)

45
Q

If acceptable, label slide as follows:

A

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

Hematology (12 decks)

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