WBC Differential Count

WBC Differential Count

• Last test for Complete Blood Count

• Why perform?
• To specify “how much specific type of white cell is present in
patients blood”?, we can have an impression on what type of
infection has.
• Through differential count, though it is not confirmatory,
but at least it provides us a picture of what could be the
possible cause of infection among patients.
• Example:
• Lymphocyte = Increase = Viral infection
• Neutrophil = Increase = Bacterial Infection
Uses of Blood Smear Examination
1) WBC differential count

• You can also use it for WBC Counting, particularly

WBC counting estimate/WBC estimate, but NOT
RECOMMENDED because the distribution of cells will
VARY from one smear to another

• Example:
• Thick Smear = HIGH cell count
• Thick smear → lot of blood → lot of cells
• Thin smear = LOW cell count
2) Platelet Examination

• Example:
• Blood specimen is tested using a Automated Machine and it
revealed the Platelet count is LOW.
• What you do is: TEST THE SAME SPECIMEN USING THE
SAME MACHINE. If the result is still LOW , then prepare a
BLOOD SMEAR.
• Blood Smear: Check if platelets are DECREASED in the
distribution of patients blood and check if there are
abnormalities in patients blood that could affect platelet
count.
3) Morphological evaluation of formed elements

• IMPORTANT: If you will be examining a smear, let’s say you only

want Diff count, you should also be mindful of the characteristics
of other cells within the smear. You need to be mindful of the
morphologic characteristics of other cells.
• Blood smear gives us a picture of the true condition of our
blood. Whatever size, whatever shape, whatever appearance of
your cells are, it could only be picture out by examining a smear.
• It is the picture of the true condition of our blood cells: Blood
smear.
• Any morphologic changes, normal morphology, abnormal
morphology, abnormal cells, it could be identified if you will examine
a smear
Specimen for Smear Examination
• EDTA blood
as much as possible use LIQUID EDTA
• ADVANTAGES:
• It preserves the morphology features of the cells. Whatever
size or shape.

• Prevents the clotting/clumping of platelets.

• Platelets are attracted to negative charge and glass
surfaces have NEGATIVE CHARGE.
• Without anticoagulant = platelets may aggregate on the
slide

• Can prepare MULTIPLE smears if necessary .

• DISADVANTAGE:
• Platelet Satellitism or Platelet Satellitosis
• WBC surrounded by platelets. Because EDTA may induce certain
ANTIBODIES (an IgG antibody forms in the presence of EDTA)
that will result to the binding of platelets to your neutrophil.
• Rarely happens.
• Blood smear to know if it is present.
• FALSELY LOW platelet count
• Upon smear examination, and then you notice that there is Platelet
Satellitism, the next thing you do is collect another blood. But
instead of using EDTA, you make use of CITRATE.
• You make use of citrate only if there is Satellitism
• YOU ONLY USE CITRATE FOR PLATELET COUNTING. And if
there is Satellitism. If you don’t have Satellitism, still it would
be EDTA.
• Prepare a smear from a FRESHLY collected
specimen/blood
• Blood films in room temperature/RT for more than 5 hours often
have unacceptable artifacts

• If you prepare a smear after 5 hours from the time you have
collected the blood, there maybe certain ARTIFACTS or SOME
CELLS may already be DESTROYED (RBCs maybe damage,
crenated, spherocytic, etc.)

• After blood is collected, prepare a smear within 2-3 hours to

obtain optimum results.
Aside from EDTA, we can also use blood collected directly from the skin puncture. We can prepare
smears. Problem is that, if it is from the skin puncture, the amount of blood is limited. But let’s say,
you are required to prepare multiple slides, 100 smear for example, you cannot use skin puncture,
you can make use of EDTA as anticoagulant.

• Capillary Blood Sample

• Directly from skin puncture.
• After you wipe the first drop of blood, you can directly add
the drop of the blood on the slide and you prepare your
smear.
• Finger and heel punctures
• Limitations:
• Some platelet clumping must be expected
• Only a few films can be made directly from blood from
a skin puncture.
• Limited slides to be prepared, and at the same time,
because of the absence of anticoagulant, platelet may
then clump or aggregate within the glass slide.
• That is why we prevent / avoid the use of capillary
blood directly for blood smearing examination.
• Whether we like it or not, from that skin puncture, blood
will stop coming out later on. Because inside, you have
already formed clots.
Peripheral Blood Smear Preparation
• Types of Blood Smear Techniques:
1. Wedge Technique (double slide / spreader slide / push
smear)
• Commonly used because it is convenient.
• We use two glass slides: One serves as a reservoir, and one
serves as spreader slide.
2. Coverslip technique (Ehrlich’s method)
3. Coverslip and slide technique (Beacon’s method)
4. Automated slide making (Spunner’s method)
• We have automated analyzers that will prepare smear, and at the
same time stain the smear for you. § There are analyzers today
that can also examine the smear for you.
1. Wedge Technique (double slide / spreader slide / push smear)
• Easiest way of preparing blood smear
• The most convenient and commonly used method for making peripheral
blood films.
• One slide serves as the film slide, and the other is the pusher or spreader slide.

IMPORTANT FACTOR: Amount of Blood that is used. Because it will affect how
thick or thin your blood smears
• Blood drop: 2-3 mm in diameter and 0.25 inch away from the ends of the
slide.
• Ideally speaking, the drop of blood should be about 2-3 mm in diameter.

• Should not be too much nor too less.

• Too much blood will result to thicker smear, thicker smear will result higher cell
count.
• Less than 2 will mean thinner smear, thinner smear will mean lesser count.
Diff-Safe Dispenser
Imagine the EDTA Tube, meron siyang cover, the Diff-
safe dispenser is inserted directly on the cap of the EDTA
tube and it will then be used to add the drop of blood to
your slide. Para hindi siya sobrang dami, and hindi siya
sobrang konti.

To make sure that you will place enough amount of blood

to your slide.

Approximately 2-3 mm diameter

Automated Machines
• The amount of blood to be used is based on the
HEMATOCRIT of the patient.
• HIGH HEMATOCRIT = HAVE A LOT OF RBCS

•INCREASED HEMATOCRIT = LESS

BLOOD IS USED
•LOW HEMATOCRIT = MORE BLOOD IS
USED
FOUR FACTORS THAT AFFECT THE SMEAR
Thick Smear Thin Smear
Pressure ↓ ↑
DECREASED/slightl INCREASED in
y touching the blood pressure
Angle ↑ ↓
> 45 º or almost 90 º < 30 º
Speed ↑ ↓
INCREASED speed DECREASED speed
Specimen (amount ↑ ↓
of blood) MORE BLOOD LESS BLOOD
*AMOUNT OF BLOOD is the MOST IMPORTANT
FACTOR
FOUR FACTORS THAT AFFECT THE SMEAR
• Pressure – amount of pressure apply in the drop of blood, as
your spreader slide comes in contact with your slide pressure is
applied.
• Spreader slide is MERELY/SLIGHTLY touching the blood = THICK SMEAR
• Spreader slide is touching the blood TOO MUCH = THIN SMEAR

• Angle
• Ideally: The
angle in between spreader slide and
drop of blood is 30 – 45 º
• If the angle is HIGHER than 30 – 45 º or almost 90 º =
THICK SMEAR
• If the angle is LOWER than 30 º = THIN SMEAR
FOUR FACTORS THAT AFFECT THE SMEAR
• Speed
• Not to fast and not to slow, just right
• Spread to quickly = SHORTER LENGTH, DARK RED in
COLOR, and JAGGED ENDS
• Spread to SLOW = THIN SMEAR

• Specimen (amount of blood)

• MORE BLOOD = THICK SMEAR
• LESS BLOOD = THIN SMEAR
TOUNGUE shaped, no holes, no bubbles, should not be touching the
edge of slide (Because WBCs may accumulate in the edge), and
should have a TRANSITION OF COLORS (dark (thick) to light (light)),
OMBRE.
A. Chipped or rough edge on the spreader slide.
B. Hesitation in forward motion of spreader slide.
C. Spreader slide pushed too quickly
D. Drop of blood too small
E. Drop of blood not allowed to spread across the
width of the slide.
F. Dirt or grease on the slide; may also be due to
elevated lipids in the blood specimen
G. Uneven pressure on the spreader slide
H. Time delay; drop of blood began to dry.
Feature of a well-made wedge peripheral blood film
1. The film is two thirds to three fourths the length of the slide.
2. The film is finger shaped, very slightly rounded at the feather
edge, not bullet shaped; this provides the widest area for
examination.
3. The lateral edges of the film are visible.
4. The film is smooth without irregularities, holes, or streaks.
5. When the slide is held up to the light, the thin portion (feather
edge) of the film has a “rainbow” appearance.
6. The whole drop of blood is picked up and spread, shows
unacceptable films.

At the end of the day, what’s important is that, within the

smear there is a transition from thick going into the thin area.
Automated Slide Making and Staining
Staining of smears
• After you prepare your smear, the next thing you need to do is to
stain them. Because if you want to differentiate white cells, we
based it on their staining characteristics.
• Because there are acidic and basic components of cells. That is
why in staining smears, you will then be using polychromatic
stains or polychrome stains.
• Polychrome stains which is a mixture of (EOSIN and
METHYLENE BLUE) acidic and alkaline stain. Again, cells,
particularly white cells, have those acidic and alkaline
characteristics. That is why we make use of polychrome stains.
• Staining of smear is pH dependent, because cell contains
enzymes or granules that reacts best at different pH
• We stain them so we can easily identify the morphologic features of
the cells. You always check the nucleus, and the color of granules.
• Example
• 3-5 lobes with rose-pink granules: neutrophils
• very large cell with kidney bean-shaped nucleus containing
vacuoles on the cytoplasm: monocyte
• Bilobed with red-orange granules: eosinophil
• You cannot clearly see the nucleus because it is covered with
darkly stained purplish granules: Basophil
• Almost the same size as with the red cell containing a very large
nucleus almost occupying the entire cell: lymphocyte
• A cell with a S-shaped or sausage shaped nucleus: band cell (still
included in differential counting)
• Pure Wright Stain
• Wright-Giemsa Stain

• Purpose
• Make the cells more visible and to allow their morphology to be evaluated.
• Fixative
• Methanol
• Before you stain your smear, you need to make sure that you fixed your smear
first with methanol, so that cells are affix on your slide.
• pH: 6.4 – 6.8
• And since, staining reactions of your cells are pH dependent, you will need to
make use of buffers like the following:
• 0.05 M Sodium Phosphate buffer (6.4)
• Aged distilled water (6.4 – 6.8)
• So you can then balance, the pH staining reactions of your cells
• Free methylene blue is basic and stains acidic (and basophilic)
cellular components, such as ribonucleic acid (RNA)
• Free eosin is acidic and stains basic (and eosinophilic)
components, such as hemoglobin and eosinophilic granules.
• Example: eosinophils are called as such because of its acidic content.
You call it basophil because of affinity with basic stain.
• Neutrophils are called such because they are neutral.

• After you fix it with methanol, you flood it with the stain.
• Or you dip it in your copline jar.
• For some, finaflood, you stain it for 2-3 minutes, 5 minutes at some stains,
then yung iba sinasawsaw lang.
• What’s important: before you stain it, you need to make
sure first that you are able to fix them with methanol.
 Prepare a Fix with Dip 2-3 times in
Dry the smear methanol
smear METHANOL

Dry Stain Buffer

Dip 6-8 times in Dip 3-4 times in
stain buffer

DON’T USE TAP Wash with Microscopic

Dry the smear
WATER distilled water analysis
• Well-Stained Blood Smear
• Macroscopically
• Reddish brown: no stain Violet: Eosin and Meth Blue
• Wright’s stain (pink)
• It may not be enough, but microscopically that is how we can fully confirm if
we have really stained the smear properly or not. Specially we check the
color of the red cells. Hindi naman siya red, may pagka-pinkish siya ng konti.
• You check the color of the white cells nucleus, purplish in color.
• Microscopically: Minimum precipitate with no artifacts
• RBC: Pink
• Nuclei of Leukocytes: Purple
• Eosinophilic granules: Red orange
• Basophilic granules: Dark purple
• Platelets: Dark Lilac
• Monocytes: Gray ground glass appearance
Staining Problems
TOO ACIDIC STAIN TOO ALKALINE STAIN
Thin blood smear Thick blood smear
Insufficient staining time Prolonged staining
Prolonged buffering or Insufficient washing
washing
Old stain Alkaline pH of stain
components
Correction: Correction:
Lengthen staining time Check pH
Check stain and buffer pH Shorten stain time
• For example, the blood smear is thin, there is
insufficient staining time, prolonged buffering, what
can we do is, tagalan yung staining if manipis
yung smear.
• Manipis na yung smear, tagalan yung staining so
you can then make sure that the cells will take up
the stain.
• Check the pH time to time if it is still buffered or
not.
• Pag baliktad naman, makapal yung smear,
bilisan yung pag- stain.
Troubleshooting Poorly Stained Blood Films
First Scenario
Problems
• RBCs appear GRAY Check pH
• WBCs are too DARK
• Eosinophil granules are GRAY, not ORANGE

Causes
• Stain or buffer too alkaline (most common)
• Inadequate rinsing
• Prolonged staining
• Heparinized blood sample
• Thick smear
Troubleshooting Poorly Stained Blood Films
Second Scenario
Problems
• RBCs are too PALE or are RED
• WBCs are BARELY VISIBLE

Causes
• Stain or buffer too ACIDIC (most common)
• UNDERBUFFERING
• OVER-RINSING
• HEPARINIZED BLOOD – remember, we don’t use
heparin as anticoagulant for blood smear preparation.
Because heparin imparts a bluish discoloration on your
smear, on the background of your smear. Having this
colored background may then result to a problem in the
identification of your cells.
• If the background is also colored, it may then interfere
with the identification of the cells. It would be a lot more
difficultfor you to identify your cells
Macroscopic Examination
• Bluer than normal: increased blood proteins and
possible rouleaux
• Dark yung color niya, it could then be associated with
elevation on plasma proteins, there is rouleaux
formation. Or you can associate it with diseases
related to increase antibodies. Increased
immunoglobulins like in multiple myeloma. Flame cell
is seen
• Grainy: RBC agglutination
• Example: associated with cold agglutinins or primary
typical pneumonia
• Mix well EDTA before smear
• What is that antibody associated with autoimmune
hemolytic anemia with cold antibodies resulting to cold
agglutination? Anti-i, with mycoplasma pneumonia
• Having such antibody may result agglutination of red cells. Macroscopically on
the smear, pwedeng meron parang mga grains.
• Holes all over the smear: Increased lipid levels/too diluted
specimen or blood mixed with water
• If there is a lot of holes all over the smear, it could be because of
increased lipids.
• Blue specks at the feathery edge: Markedly increased
WBC and platelet counts
• Nagkukumpulan na yung WBC, kaya hangga’t kaya, the end of the
blood on your smear should not reached the end of the slide or do
not touch the end of the slide.
Microscopic Examination
• Microscopically, you always follow PARFOCAL
FOCUSING
• Scanner → LPO → HPO → OIO, with very little
adjustments
Low Power Objective (LPO / 10X)
Overall film quality
• Under scanner LPO, either you can check the overall quality of
your smear or blood film.
• You can check whether your smear is acceptable or not
Color
• You can check the color, masyado bang gray yung RBC,
masyado bang dark yung WBCs, and the likes.
Distribution of cells
• Check for overlapping cells or clumping cells
• Check for Rouleaux formation
• Generally, to know if the smear is good for examination
or not, you can go with LPO
• Possible to check for the presence of fibrin strands
• RBC distribution or RBC agglutinins
• Scanned quickly for any large abnormal cells
• Feather edge and lateral edges should be checked
quickly for WBC distribution
High Power Objective (HPO / 40X)
• Select the correct area of the film in which to begin the
differential count
• Cells should be equally distributed
• You should select on the part where cells are not overlapping.
• Evaluate cellular morphology
• WBC Estimate
• Under HPO, this is where you can count WBCs. Again that is an
estimate. In counting white cells, under HPO, you just need to count 10
HPO/HPF
• After counting all white cells in 10 HPF, simply get the average.
• So to compute for your WBC Count, your average multiplied by 2,000,
that is now your WBC count.
• Average x 2,000 = WBC Count (estimate)
Oil Immersion Objective (OIO / 100X)
• Add the oil between HPO and OIO
• Provides the highest magnification
• WBC Differential count
• Since OIO provides the highest magnification,
remember that differential counting is done at
OIO.
• RBC, WBC, and Platelet morphology evaluation
• Checking the morphology of cells is done at OIO.
• If you want to if there would be any abnormalities,
poikilocytes, inclusion bodies, abnormal white
cells, it can only be identified under OIO.
• Platelet estimate
• This is where you also count your platelets.
• WBCs counted using HPO, but for platelets its
under OIO.
• count at least 10 OIO fields for platelet estimate
and then get the average, after you get the
average, multiply it by 20,000
• Average x 20,000 = Platelet estimate
• the number of cells per microliter (uL)
• To convert it to SI unit, both WBC count and
Platelet count, will be multiplied by 0.001.
•And both WBC count and Platelet count
9
should be reported as x10 /L
•Segmented neutrophils can be readily
differentiated from bands
•RBC and WBC inclusions
•Reactive or abnormal cells enumeration
•N-RBC counting (if present)
•check if there are nucleated red cells, still,
you can identify it under OIO.
Optimal Assessment Area
How are we supposed to count the cells?
• Between thick area and the very thin feather edge
• Ideal
• RBC are uniformly and singly distributed, with few touching or
overlapping, and have their normal biconcave appearance
• 200 to 250 RBCs per Oil Immersion Field
• You need to select first on that site where cells are equally
distributed, they are not overlapping, and normally under
OIO, every OIO will contain about 200 to 250 RBCs, normally.
• If that is the case, then it tells you that you are in the right
field
Counting Methods
• DON’T go back to the SAME area
• DON’T go back to the SAME field

BATTLEMENT TECHNIQUE – most commonly used

• Zizag like - not going back on the field that you have already
counted.
• You follow battlement technique, but still making sure that
you are on the part where cells are equally distributed.
• Cross-sectional or Crenellation: from side to side
• Longitudinal: from the tail toward the head of the smear
• Battlement: uses a pattern of consecutive fields beginning near
the tail on a horizontal edge
Differential Count
Principle: A stained smear is examined to
determine the percentage of each type of
leukocyte present and assess the erythrocyte
and platelet morphology
• Specimen
• Peripheral blood
• Bone marrow
• Body fluid sediments
Differential Count
• Routinely = 100 WBCs
• count and differentiate them manually.
Example
• WBC count is 40 x109/L – HIGH = 200 WBCs are to be differentiated to
increase ACCURACY
• 100 x109/L = 300 – 400 WBCs is counted
The more the patients white cell count increases, the more white
cells are to be differentiated.
• One hundred WBCs are counted and classified through the use
of push-down button counters
• Results are reported as percentages
•WBC Abnormalities are also reported
• Reporting:
• Reactive Lymphocytes
• Should be reported semi-quantitatively
• Separate
• Semi-quantitatively; you report it as occasional to
many
• Sometimes, it can also be reported as 1+, 2+, 3+
• Toxic granulation
• Present
• Semi-quantitatively: slight to marked / 1+ to 3+
 A Comparison of Normal Leukocytes in Peripheral Blood

Segmented Band Lymphocyte Monocyte Eosinophil Basophil

Neutrophil Neutrophil

Nuclear Lobulated Curved Round Indented or Lobulated Lobulated

Shape twisted

Chromatin very Moderately Smooth Lacy Very Very

clumped clumped clumped clumped

Cytoplasmi Pink Blue, pink Light blue Gray-blue Granulated Granulated

c color

Granules Many Many Few or Many Many Many

absent

Color of Pink, a few Pink Red Dusty blue Orange Dark blue
granules blue

Average 56% 3 % 34% 4% 2.7% 0.3%

percentage

NEVER LET MONKEYS EAT BANANAS Band CELL = S-shaped nucleus included in count
Procedure
1. Begin the slide examination with a correctly prepared and stained
smear.
2. Focus the microscope on the 10x objective (low power). Scan the
smear to check for cell distribution, clumping, and abnormal cells.
Add a drop of immersion oil and switch to the 100x (oil
immersion) objective. Begin the count by determining a suitable
area. Extend the examination from the area where approximately
half of the erythrocytes are barely overlapping to an area where
the erythrocytes touch each other. It is important to examine
cellular morphology and to count leukocytes in areas that are
neither too thick nor too thin. In areas that are too thick, cellular
details such as nuclear chromatin patterns are difficult to
examine. In areas that are too thin, distortion of cells makes it
risky to identify a cell type.
3. Count the leukocytes using a tracking pattern. Each cell identified
should be immediately tallied as a neutrophil (band), neutrophil
(segmented), or polymorphonuclear neutrophil (PMN); lymphocyte;
monocyte; eosinophil; or basophil. A brief leukocyte morphology
reference is included; however, refer to specific chapters in the text for a
complete discussion of leukocyte and erythrocyte cellular morphology.
4. Abnormalities of leukocytes, erythrocytes, and platelets should be
noted. Normally, 8 to 20 platelets are present in an oil immersion field in
a properly prepared smear (where the RBCs barely touch each other).
After examining at least 10 different fields, the average number of
platelets can be multiplied by a factor of 20,000 to arrive at an
approximate total circulating platelet concentration. Nucleated
erythrocytes are not included in the total count but are noted per 100
white blood cells (WBCs). A total of at least 100 leukocytes should be
counted. Express the results as a percentage of total leukocytes
counted.
Absolute Count/ Absolute WBC Differential Count
• more accurate than relative count
• the actual number of specific WBC in a Liter of blood
• ABSOLUTE = TRUE VALUE

• Represent the number of specific type of WBC for every liter of blood
(kada liter ng blood di lang 100 WBCs ang present you have more than
that)
• With your differential count > out of 100 white cells, whatever value or
results you have will just be considered as RELATIVE COUNT
- does not represent entirety of WBC because it is only a representation
of 100 white cells > so a lot of clinicians will request more for
ABSOLUTE COUNT OR TRUE VALUE
FORMULA: Percentage x WBC Count

• COMPUTATION: UNIT: x 109/L

• If u will add the total of your ABSOLUTE DIFFERENTIAL

COUNT, Dapat equal sila lahat sa total ng patient’s WBC
count
Example:
• WBC Count = 7 x109/L
• Diff count: out of 100 cells, 50% are neutrophils, 35% are
lymphocytes, 10% are monocytes, 3% are eosinophils, and 2% are
basophils

• To compute:
• 50% x 10 = 3.5 x109/L meaning, out of 10, 5 of them are
neutrophils. And take note, if you will add all absolute count, it
needs to be equivalent to your WBC count. Pag in-add mo yan
dapat equal to 10.
• 50% x 10 = 5 x109/L
• 30% x 10 = 3 x109/L
• 10% x 10 = 1 x109/L
• 5% x 10 = 0.5 x109/L
• 5% x 10 = 0.5 x109/L