PBS AND DIFFERENTIAL COUNT Flashcards
Materials used:-
Glass slide
-Spreader
-Blood sample (whole blood)
WEDGE BLOOD SMEARS
-Place a drop of blood 1cm away from labelled end
-When using blood from finger or heel, prevent touching the skin with the slide
-Hold the slide with blood in one hand and the spreader on the other hand
-Place the spreader slightly in front of the blood drop (30-40 degree angle)
-Draw the spreader slide back
-As soon as blood comes in contact with spreader, it begins to spread to the edges, if not, wiggle it a little
-Push the spreader rapidly over the entire length of the slide
Let dry, stain.
a) Glass slides must be clean
WEDGE BLOOD SMEARS
b) As soon as blood is placed on the slide, immediately make the smear
WEDGE BLOOD SMEARS
WEDGE BLOOD SMEARS delay
abnormal distribution of wbc (many large cells accumulate at the thin edge) rouleaux of red cells and plt clumping.
WEDGE BLOOD SMEARS
Causes of poor blood smear:
-drop of blood too large or too small-Spreader slide pushed in a jerky manner
-Failure to put the entire edge of the spreader slide against the slide while making the smear
-Failure to put the spreader in a proper angle against the slide.
-Failure to push the spreader slide completely across the slide
results in thicker smear for very low hct
Increase angle
gives a thin smear for patients with very high hematocrit
Decrease angle
Too much Drop of blood
Too little Drop of blood
Angle Too high
Angle Too low
Pressure Decreased
Pressure Increased
Speed Too slow
Speed Too fast
A. Materials used:
-2 cover slips (22mm thick, 0.13-0.17 mm thick)
-Blood sample
COVER GLASS SMEARS
COVER GLASS SMEARS
B. Procedure:
-Hold 1 cover glass by its [?], place a drop of blood
2 adjacent corners
COVER GLASS SMEARS
B. Procedure:
-With the second hand, hold the [?] as the first
second cover slip
COVER GLASS SMEARS
B. Procedure:
-Gently place the [?] on top of the one containing blood
second cover glass
COVER GLASS SMEARS
B. Procedure:
drop of blood in the middle of the 2 glass slides forming
16 sides
COVER GLASS SMEARS
B. Procedure:
-Just before spreading of blood is complete, separate the 2 cover glasses by a [?] (avoid squeezing the 2 glass slides together)
rapid, even, horizontal, lateral pull
COVER GLASS SMEARS
B. Procedure:
-Allow the smears to [?]
-Stain
air dry completely
Cover glasses must be clean
When obtaining blood from finger tip puncture, do not touch the skin
The cover glasses should be put together without delay upon dropping of blood
COVER GLASS SMEARS delay
clumping of plts and wbcs, rouleaux formation
-Too large drop of blood causes too thick smear
COVER GLASS SMEARS
-This type of smear prep gives a more even distribution of wbcs than the wedge
COVER GLASS SMEARS
More time consuming, tedious, harder to master
COVER GLASS SMEARS
Harder to label
COVER GLASS SMEARS
Coverslips are easily broken
COVER GLASS SMEARS
A. Materials used
-Blood sample
-Instrument: Hemaspinner
AUTOMATED SPUN SMEAR
AUTOMATED SPUN SMEAR
B. Procedure
-A clean glass slide is placed on a plate, [?] are placed at the center of the slide.
3-4 drops of blood
AUTOMATED SPUN SMEAR
B. Procedure
-When the top of the instrument is closed, the plate spins at a high speed for a period of time (excess blood is thrown from the slide to a basin)
AUTOMATED SPUN SMEAR
B. Procedure
-Resultant slide is completely covered with a [?]
-Stain
monolayer of cells
A. Materials used:
-Wintrobe tube
-Capillary pipette, steel needle
-Glass slides-Coverslip
BUFFY COAT SMEAR
BUFFY COAT SMEAR
B. Procedure:
-Using the capillary pipette, fill a [?] with well mixed whole blood
wintrobe tube
BUFFY COAT SMEAR
B. Procedure:
-Centri:
15 minutes, 1500g
BUFFY COAT SMEAR
B. Procedure:
Locate the buffy coat, separate [?] except a small amount near the buffy coat
plasma
BUFFY COAT SMEAR
B. Procedure:
-Using capillary pipette, remove the
small amount of remaining plasma, the entire buffy coat, small amount of red cells
BUFFY COAT SMEAR
B. Procedure:
-Place on a glass slide, [?] (do not spread out too much on the slide)
mix
BUFFY COAT SMEAR
B. Procedure:
-Transfer a small amount of blood to each of two slides, immediately prepare
wedge or cover and a glass smear
BUFFY COAT SMEAR
The amount of [?] should be approximately equal to the volume of the cellular portion of the mixture
plasma mixed with the buffy coat and red cells
Microhematocrit tubes may also be used to centrifuge the specimen
BUFFY COAT SMEAR
cut the tube [?], transfer blood onto slide
near the buffy coat layer
[?] on a buffy coat smear does not always make an accurate differential count
Distribution of WBCs
tend to settle according to their cell type during the centrifugation process
nucleated cells
the differentials from a well made buffy coat smear correlate relatively well with the standard blood smear
leukopenia
A. Materials used:
-Blood sample
-Glasss slide
THICK BLOOD SMEAR
THICK BLOOD SMEAR
B. Procedure
Place [?] in the center of a glass slide
one large drop of blood
THICK BLOOD SMEAR
B. Procedure
Using the [?], carefully spread the drop of blood over an area the size of a dime
corner of the second slide
THICK BLOOD SMEAR
B. Procedure
[?]: place newspaper behind the slide.
Spread the drop od blood until the newspaper print is just visible through the blood.
Thickness
THICK BLOOD SMEAR
B. Procedure
Allow the film to completely dry before staining (?)
4-2 hrs at room temp, preferably overnight
THICK BLOOD SMEAR
B. Procedure
-If not completely dry, will [?] during staining. (This type of stain is not fixed prior to staining)
wash off
Stains used:
Romanowsky Stains
-Wright’s (oxidized methylene blue, eosin azures)
-Wright’s
– Giemsa/ Modified (more delicate staining char)
-Leishman
-Jenner
-May-Gruwald
(oxidized methylene blue, eosin azures)
-Wright’s
(more delicate staining char)
-Wright’s– Giemsa/ Modified
oxidized methylene blue
Eosin B or Eosin Y
oxidized methylene blue products
Azure B
Are considered polychromatic (Impart different colors to cells and cellular elements)
Romanowsky Stains
promotes ionization during which time staining takes place
Buffer
neg charged, azure B: pos charged
eosin ions
STAINING
Procedure:
-Place the dried smear level on a [?]
-Fix by flooding with methanol-Flood slides with [?], time for 4 minutes
-Without removing the stain, add equal volume of [?]
-Mix 2 solutions on the slide by gently blowing back and forth over the solutions (a [?] should form, time for 7 minutes)-rinse thoroughly with tap water
-Wipe the [?], air dry
staining rack
Wright-Giemsa stains
phosphate buffer
metallic green sheen
back of the slides
a) Pink to orange
red blood cells
b) Pinkish-grey
reticulocytes
c) Dark purple nuclei
lymphocytes and neutrophils
d) Lighter nucleus
monocytes
e) Bright orange granules
eosinophils
f) Dark blue black granules
basophils
g) Violet to purple granules
platelet
h) gray blue with fine reddish granules
Cytoplasm of the monocyte
has a light pink cytoplasm with lilac granules
Neutrophil
shows varying shades of blue cytoplasm
Lymphocyte
cell parts taking acidic dyes appear more pink, parts taking basic dye show pale staining
too acidic
rbcs appear greyish-blue, white cell uclei stain deeply purple
too alkaline
equal staining of the film
Staining rack should be level
causes stain to precipitate on the dried smear
Insufficient washing
fades the stain
Excessive rinsing
If restain: flood smear with [?] and rinse to remove the stain BUT, new smear and stain is always advisable.
methanol
For [?], after staining is done, mount the cover slip blood side down onto a glass slide with mounting medium.
coverglass smears
Stock solutions of individual stains must be freshly prepared how often?
each week
Staining buffer solution may only be used for
1 hour
controls pH
Phosphate buffer
Performed to determine the relative number of each type of white blood cell present in the blood.
DIFFERENTIAL COUNT
-Study of red cell, white blood cell and platelet morphology is performed
DIFFERENTIAL COUNT
Approximation of the number of platelets are made
DIFFERENTIAL COUNT
-Performed after the cell counts have been completed
DIFFERENTIAL COUNT
-May be used to double check the wbc count
DIFFERENTIAL COUNT
tend to show increased concentrations of PMNs, monocytes and large abnormal cells at the edges and tail od the smear
Too thin smears
causes increased lymphocytes at the center of the smear
Too thin smears
a. Appendicitis
Neutrophilia
b. Myelogenous leukemia
Neutrophilia
c. Bacterial infections
Neutrophilia
Eosinophilia
a. Allergies, allergic reactions
b. Scarlet fever
c. Parasitic infections
d. Eosinophilic leukemia
absolute increase in neutrophil count
Neutrophilia
Lymphocytosis
a. Viral infections
b. Whooping cough
c. Infectious mononucleosis
d. Lymphocytic leukemia
Monocytosis
a. Brucellosis
b. Tuberculosis
c. Monocytic leukemia
d. SBE
e. Typhoid
f. Rickettsial inf.
DIFFERENTIAL COUNT PROCEDURE:
a) Place the prepared smear prep on the stage of the microscope
b) Examine under [?]
c) Take note of anything [?]
d) Examine under [?]
LPO
unusual or irregular
HPO
DIFFERENTIAL COUNT PROCEDURE:
Examine under LPO
a) Check for [?] of white blood cells
b) Examine the [?]
c) If there is an [?] in this area, the diff count may be inaccurate
d) If there are [?], the body of the smear may show decreased plt count
e) Discard smear and make a new one
even distribution
thin peripheral edge
increased number of white cells
plt clumps
Take note of anything unusual or irregular
a) Large abnormal looking cells, rouleaux formation
Examine under HPO
WBC estimate
(average WBC in 10 fields)
DIFFERENTIAL COUNT PROCEDURES
a) Cross sectional, crenellation
b) Lonhitudinal
c) Battlement
[?] each white blood cell seen and [?] in a differential counter until 100 cells have been counted.
Identify; record
a) If any [?] are seen, record on a separate sheet of pad paper
h) Examine red cell morphology in a [?] where small amount of red cells only slightly overlap
a) Note variations from normal, classify irregularities as [?]
nucleated red cells
thin area
slight, moderate, marked
Examine plts for number and morphology
a) Determine approximate number of plts per field [?]
b) plt estimate = [?]
(8-20 plts per field)
average number of plts in 10 OIO fields x 20,000
Results are reported as
percentage of the diff wbc cell types
The most preferred method of reporting = absolute wbc count
Absolute # of cells/L = % of cell type in diff x WBC/L
c) Do not progress too far into the [?] bec morphology is difficult to observe here.
thick area
d) Do not use the [?] as the red cells appear to have no central pallor area
very thin area
When WBC count is below 1.0x10^9/L, it may be difficult to find WBCs on the stained smear
May perform a 50 differential (NOTE it), buffy coat smear may be prepared
If abnormal distribution of cell types:
200 cell differential may be done, results divided by 2 (NOTE it)
If the diff count shows immature granulocytic cells
shift to the left = [?]
shift to the right = [?]
leukemia
increased numbers of hypersegmented neutrophils
Smear shows (?).
The white blood cell count is (?).
The differential count is as follows: [?].
No [?] seen.
Platelets are (?).
Smear is (?), (?).
red cell morphology
Decreased/WTNR/Increased
Neutrophils: %, Lymphocytes: %, Monocytes: %, Eosinophils: %, Basophils: %
atypical cells
decreased, adequate, increased
positive/negative for malarial parasites
Genus, species, stage of development
