GHOR ALSAFI LAB.

Differential Count

A.KAREEM AL-AJOURI 1

White Blood Cells
Differential Count



1. Introduction:

To small laboratories, white blood cell differential becomes a high complexity test
if:
not performed as a direct readout from an automated analyzer
and / or atypical cells are identified.
By including the white blood cell differential in the test menu, the laboratory will
need:
Increased level of necessary staff training
Quality control
Continuing education
An efficient strategy would be to refer blood specimens outside when the white
blood cell count is abnormal initially or significantly changed.

2. Definition:

The white blood cell differential count is performed to determine the relative
number of each type of white blood cell present in the blood.





3. The Normal Reference Ranges:

3.1. Normal Ranges for WBC Differential Count; values expressed in percentages:

3.1. Normal Ranges for WBC Differential Count; values expressed in percentages:
Age
Neutrophils Lymphocytes Monocytes
Neonates & Children
10% - 25% 75% 90% 0% 8%
Adults 25% 75 % 25% 60% 0% 8%



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A.KAREEM AL-AJOURI 2

3.2. Normal Ranges for WBC Differential Count; values expressed cells X 10
9
/L:

Table 3.2 Normal Ranges for WBC Differential Count (cells X 10
9
/L)
Age
Neutrophils
Eosinophils Basophils Lympho-
cytes
Mono-
cytes
Total
WBC
Count
Total Band Segmented
Birth
6.0-
26
1.0-
1.6
9-11 0.02-0.85 0-0.64 2.0-11.0 0.4-3.1 9-30
7 Days 1.5-
10.0
0-
0.83
2.0-4.7 0.07-1.1 0-0.25 2.0-17.0 0.3-2.7 5.0-
21.0
14
Days
1.0-
9.5
0-
0.63
0-3.9 0.07-1.0 0-0.23 2.0-17.0 0.2-2.4 5.0-
20.0
12
Month
1.5-
8.5
3 1.0- 8.5 0.05- 0.70 0.0- 10.5 4.0-10.5 6.0-
17.5
4
Years
1.5-
8.5
0- 1.0 1.5-7.5 0.02-0.65 0.0-2.0 2.0-8.0 0-0.8 5.5-
15.5
6
Years
1.5-
8.0
0-10 1.5-7.0 0-0.65 0-0.2 1.5-7.0 0-0.8 5.0-
14.5
10
Years
1.8-
8.0
0-1.0 1.8-7.0 0-0.6 0-0.2 1.5-6.5 0-0.8 4.5-
13.5
21
Years
&
above
1.8-
7.7
0-0.7 1.8-7.0 0-0.45 0-0.2 1.0-4.8 0-0.08 4.5-
11.0


4. Clinical Significance:

In certain disease states, a particular white blood cell type may show an absolute
increase in number in the blood.
Common diseases showing an increased number of a specified cell type are listed
bellow:
Absolute increase in the number of neutrophils (neutrophilia):
o Appendicitis
o Myelogenous leukemia
o Bacterial infection
Absolute increase in the number of eosinophils (eosinphilia):
o Allergies
o Scarlet fever
o Parasitic infection
o Eosinophilic leukemia
Absolute increase in the number of basophils (basophilia):
o Allergic reactions
o Chronic myelogenous leukemia
o Basophilic leukemia
o Polycythemia vera
o Following irradiation (transient basophilia)
GHOR ALSAFI LAB. Differential Count

A.KAREEM AL-AJOURI 3
Absolute increase in the number of lymphocytes (lymphocytosis):
o Viral infection
o Whooping cough
o Infectious mononucleosis
o Lymphocytic leukemia
Absolute increase in the number of monocytes (monocytosis):
o Brucellosis
o Tuberculosis
o Monocytic leukemia
o Subacute bacterial endocarditis
o Typhoid
o Ricketsial disease
o Hodgkins disease
o Gauchers disease

5. Preanalytical Conditions:

5.1. Preparation of Patient:

Child and Adult: no special procedure to follow.

5.2. Collection of Specimen:

Morning rested state collection of EDTA blood is recommended.

5.3. Storage Considerations:

Sample is stored at room temperature and is analyzed within 4 hours.

6. Reagents:

6.1 Preparation of the Wright Stain:

6.1.1 Mix the following in a large, tightly stoppered brown bottle:


Wright stain powder 10 grams
Glycerin 90 mL
Methanol 2,910 mL

6.1.2. The stain should be allowed to age for approximately 30 days prior to use. During this
time the stain should be shaken once a day. Incubation at 37C speeds the aging process.
6.1.3. The stain must be freshly filtered before use (only filter 1- or 2- day supply at a time).
GHOR ALSAFI LAB. Differential Count

A.KAREEM AL-AJOURI 4
6.1.4. After the stain is ready to use, use it for a maximum of one month, then discard and
prepare a fresh stain.

6.2. Preparation of the Phosphate Buffer

6.2.1. Mix the following in a large, volumetric flask:
Anhydrous monobasic potassium phosphate (KH2PO4) 6.63 grams
Anhydrous dibasic sodium phosphate (Na2HPO4) 2.56 grams
Distilled water 1000 mL

6.2.2. The pH of the buffer solution should be within a pH range of 6.4 to 6.7, depending on
the staining times and the type of Wright stain used.

7. Procedure:

Table 7. Procedure for Blood Films
Step Procedure

7.1. Manual preparation of the blood smear:
7.1.1. Obtain two clean and dust-free glass slides and a microhematocrit tube.
If using anticoagulated blood, use a plain microhematocrit tube.
One of the slides will be used as a spreader slide. The spreader slide is a glass slide with
specially ground ends to ensure even spreading of the blood. The spreader slide should
be clean and dry.
7.1.2. Fill the microhematocrit tube with the anticoagulated blood.
Carefully place a small drop of blood (2-3 mm in diameter) about 1 cm from the end of
the slide.
7.1.3. Place the slide on a flat table top with the drop of blood on the right.
For left-handed people, the technique is reversed to the opposite hand.
7.1.4. With the thumb and forefinger of the left hand, hold the two left edges of the slide. With
the right hand, hold the spreader slide with the thumb on the edge of one side end and
the forefinger on the edge of the other side. Place the end of the spreader slide slightly
in front of the drop of blood on the other slide, at an angle of 30
0
C to 45
0
C between the
two slides.
7.1.5. Draw the spreader slide back toward the drop of blood. As soon as the spreader slide
comes in contact with the drop of blood, the blood begins to spread to the edge of the
spreader slide. If this does not occur, wiggle the spreader slide a little until it does so. Be
careful that no blood gets in front of the slide.
7.1.6. Keeping the spreader slide at a 30
0
C to 45
0
C angle and the edge of the spreader slide
firmly against the horizontal slide, push the spreader slide at a moderate speed forward
until all the blood has been spread into a moderately thin film.

7.1.7. The blood smears should be dried quickly by waving them rapidly in the air; this
prevents distortion of the red blood cells. The blood smears are now ready for Wrights
stain.
7.1.8. The slide may be labeled by writing the identification with a lead pencil, wax pencil or
diamond pen on the frosted end or directly on the thicker end of the blood film.
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A.KAREEM AL-AJOURI 5

7.2. Staining of the prepared blood smear:
7.2.1. Place the air dried blood smears on a level staining rack, with the smear side up.
7.2.2. Fix the smears by flooding the slides with methanol for 1-2 minutes. Drain the excess
methanol off the slide. Alternatively, dip the smears into a coplin jar containing
methanol and then place the slides on the staining rack.
7.2.3. Flood the slides with the Wrights stain and time for 4 minutes.
7.2.4. Without removing the Wrights stain add an equal volume of phosphate buffer to the
slide. Mix the two reagents on the slide by gently blowing back and forth over the
solution. A metallic green sheen should now form on top of this mixture. Time 7
minutes.
7.2.5. Rinse the slide off thoroughly with a stream of tap water.
7.2.6. Wipe the back of the slide with a piece of gauze to remove any stain.
7.2.7. Allow the slides to air-dry in a tilted position. Never blot the smears dry.
7.3 Examination of the stained blood smear:
7.3.1. Examine the blood smear using the low-power (10x) objective:
The white blood cells should be evenly distributed over the smear.
Estimate the white blood cell count (by noting the number of white blood cell in relation
to the number or red blood cells).
Examine the thin peripheral edge of the smear, if there is an increased number of white
blood cells in this area, the differential cell count is inaccurate. Most of the cells at the
edge of the smear are the larger white blood cells, namely, neutrophils and monocytes.
This, therefore, shows poor distribution of white blood cell types in this area of the
smear.
7.3.2. Choose the portion of the blood smear where there is only a slight overlapping of the
red blood cells. Place a drop of oil on the slide and carefully change to the oil
immersion objective (100x).
7.3.3. Begin in the thin area of the slide where the red cells are slightly overlapping. Gradually
move the slide as shown in Figure 1. (Also see Figure 2). Count each white blood cell
seen and record on a differential cell counter until 100 white blood cells have been
counted. If any nucleated red blood cells are seen during the differential count,
enumerate them on a separate counter. These cells are not to be included in the 100-cell
differential count.
7.3.4. While counting the white blood cells, make a note of any abnormalities present in the
cells.

8. Technical Notes:

8.1. Technical Notes on Preparing the Blood Smear:

Table 8.1. Technical notes on preparing the blood smear:
Note Reason
The glass slides must be very clean Any trace of dirt will distort the blood
smear
As soon as the drop of blood is placed on the glass
slide, the smear should be made without delay
Any delay whatsoever results in an
abnormal distribution of WBCs cells, with
many of the larger WBCs accumulating at
thin edge of the smear
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A.KAREEM AL-AJOURI 6
8.1.1. Common Causes of a Poor Blood Smear:

The drop of blood too large or too small.
The spreader slide pushed across the slide in a jerky manner.
Failure to keep the entire edge of the spreader slide against the slide while making
the smear.
Failure to keep the spreader slide at a 25 angle with the slide (increasing the
angle results in a thicker smear, whereas a smaller angle gives a thin smear).
Failure to push the spreader slide completely across the slide.


8.2. Technical Notes on Staining the Blood Smear:

Table 8.2. Technical notes on staining the blood smear
Problem Reason
Distorted white blood cells Water in the Fixative (methanol). Care must be
taken to change the methanol in the coplin jar
several times a day and to keep the jar covered
when not in use because methanol readily takes
up water. You can place 100-200 mg of
anhydrous copper sulfite in the coplin jar
containing 50 mL methanol to minimize water
uptake by methanol
Uneven staining The staining rack is not exactly leveled.
Precipitate on the smear Insufficient washing of the smear when
removing the stain and buffer mixture
Stain fading - Leaving water on the smear after
rinsing
- Prolonged rinsing


8.2.1. Restaining:

If it is desirable to re-stain a slide, the original Wrights stain may be
removed with methanol. Flood the smear with methanol and rinse
with tap water as many time as necessary to remove the stain and
then re-stain the slide according to the previously described
procedure.






GHOR ALSAFI LAB. Differential Count

A.KAREEM AL-AJOURI 7
8.3. Technical Notes on Differential White Blood Count:

Table 8.3. Technical Notes on differential white blood count:
Problem
What to do
White blood count below 1,000 per L
(difficult to find many white blood cell on the
stained smear)
The differential is usually performed by
counting 50 white blood cells. A notation on
the report must then be made that only 50
white blood cells were counted
In a differential showing one or more of the
following:
- Over 10% eosinophils
- Over 3% basophils
- Over 12% monocytes
- More lymphocytes than neutrophils
200 white blood cells should be counted. The
results are then divided by 2 and a note made
on the report that 200 white blood cells were
counted
Presence of immature white blood cells - Note that there is a left shift (found in
infection and Leukemia)
- Refer the slide for consultation
- Be sure to add a comment that
Immature cells seen
- Phone the physician to bring this matter to his
attention
Presence of increased number of
hypersegmentated neutrophils
Note that there is a right shift
Technical Error Rate 10% to 15%