Hema I Chapter 8 Differential counting cell

Hematology

1. CHAPTER 8
DIFFERENTIAL WHITE CELL
COUNT

2. Objectives
After completion of this chapter, the student will be able to:
Define differential leukocyte count (DLC)
Discuss three general features to be incorporated in the DLC
Perform a 100 cell differential count according to criteria
established by the instructor
Differentiate a relative cell count from an absolute cell count
Perform a WBC and platelet estimate on a stained peripheral
blood smear

3. Objectives cont’d
Discuss the clinical implications of the differential
leukocyte count
Identify mature and immature leukocytes
Identify morphological abnormalities of leukocytes
Identify reactive lymphocytes, including clinical
situations associated with their presence
Identify possible sources of error and their remedies in
DLC
Apply QC in DLC

4. Outline

5. 8.1 Introduction
i. About Differential white cell count:
Is the enumeration of the relative proportions
(percentages) of the various types of white cells as
seen in stained blood films
Is used to determine the relative numbers of each
type of leukocyte.
A smear is examined and each time a leukocyte is seen,
it is both tabulated and classified on a special counter
The counting and classifying continues until 100 cells
have been observed

6. Diff count cont’d
The differential cell count also includes:
an evaluation of RBC morphology,
platelet morphology and numbers, and
general WBC morphology and estimation
With a manual differential count, inaccuracy or
deviation from the true count results both from:
maldistribution
misidentification of cells

7. Diff count cont’d
Maldistribution of cells
The different types of white cell are not distributed
evenly over a slide
The tail of the film contains:
 more neutrophils
fewer lymphocytes,
N.B. monocytes are fairly evenly distributed along
the length of the film
Large cells are preferentially distributed at the edges of
the film
Maldistribution of cells is aggravated:
 if a film is too thin or
if a spreader with a rough edge has been used

8. Diff count cont’d
ii. Clinical significance of Diff count
To evaluate the different leukocytes distribution in the
circulation in normal and pathological states

9. Diff count cont’d
iii. Principle of Differential white cell count
After taking blood sample, blood film is prepared, and
after staining with Romanowsky stains, 100, 200, 300,
etc., cells will be counted; then the percent distribution
of each cell is calculated
iv. Type of specimen: EDTA anticoagulated venous
or capillary blood
v. Equipment and Reagents
Microscope
Diff counter
Oil immersion

10. 8.2. Methods of Counting
Various methods of tracking over a slide have been
proposed to attempt to overcome errors due to
maldistribution:
The Longitudinal Strip Method
The Battlement Method
The Modified Battlement Method

11. A. The Longitudinal Strip Method
This is a method of tracking along the length of the film
The cells are counted using the 40x dry or 100x oil immersion objectives
in a strip running the whole length of the film until 100 cells are counted.
If all the cells are counted in such a strip, the differential totals will
approximate closely to the true differential count

12. Longitudinal strip method cont’d
Advantage and Disadvantages of the Method:
Advatage:
It compensates for maldistribution between the body and
the tail
Disadvantage
It does not compensate for maldistribution between the
centre and the edge
i.e., It does not allow for inclusion of neutrophils and
monocytes at the edges of the film

13. Longitudinal strip method cont’d
Disadv cont’d
Difficulty in identifying contracted heavily stained cells in
the thicker parts of the film (head part)
However, this problem is minimized in a well made
film and in practice brings little difference to results

14. B. The Battlement Method
Counting is performed by moving from side to side
across the width of the slide in the counting area just
behind the feather edge where the cells are separated
from one another and are free from artifacts.
Advantage
It compensates for maldistribution between the centre
and the edge
Disadvantage
It does not compensates for maldistribution between the
body and the tail
since the customary 100-cell differential count will not
cover a very large proportion of the length of the
blood film

15. C. The Modified Battlement Method
In modified battlement method, two fields are counted
close to the edge parallel to the edge of the film, then four
fields at right angles, then two fields parallel to the edge
and so on.
A modified battlement track is a compromise between the
two methods.
At least 100 cells should be counted.
If there is white cell aggregation, the maldistribution of
cells is so great that an accurate differential count is
impossible.

16. Cont..
Of the three methods
the lateral strip method appears to be the method of
choice because it averages out almost all of the
disadvantages of the two other methods
This is to be checked!!!
Multiple manual registers or electronic counters are used
for the count.

17. Diff count cont’d
Note: All elements of the blood film must be observed while
performing the differential count.
If any abnormality is seen in the smear, it should be
confirmed and reported
The following points should be checked:
Erythrocytes: size, shape, distribution, and degree of
hemoglobinization
presence of inclusion bodies
presence of nucleated red cells, if so, the total
leukocyte count must be corrected

18. Diff count cont’d
Platelets:
Estimation (10-20/HPF)
Do they look normal?
are there many giant or bizzare forms?
Leukocytes:
Mature? Immature? Atypical?
Average number of lobes? hypersegmented
neutrophils? Hyposegmentation?
Hypergranulated ones? vacuolation?
Toxic granulation, other inclusions
Estimation
etc
Hemoparasites:
malaria, borrelia, babesia, etc.

19. Diff count cont’d
vi. Procedure
Using the 10x dry objective, scan the smear
for:
Proper staining
WBC distribution
Presence of platelet clumps
Presence of light blue fibrin strands which
may indicate that the blood was not well
mixed or partially clotted
Any other unusual or irregular
characteristics of cells
Using the 40x dry objective, perform WBC
estimate (see Table below)
Platelet Clump

20. Performing a WBC Estimate Under 40x High Dry
Look for estimate area which has 200 RBCs that are
partially overlapping with many single red cells
Determine a WBC estimate using the following
conversion information (hpf=high power field):
2-4 cells/hpf = 4.0 – 7.0x10
9
/L
4-6 cells/hpf = 7.0 – 10.0x10
9
/L
6-10 cells/hpf = 10.0 – 13.0x10
9
/L
10-20 cells/hpf = 13.0 – 18.0x10
9
/L
Estimates:
are reported as decreased, Increased, or adequate
are used for QC purpose
Remember!
Estimates
are not
reported!

21. Procedure cont’d
Locate a thin area of the smear in which the RBCs
are evenly distributed, only slightly overlap, and the
central pallor or “whites-of-their-eyes” is seen
Place a drop of oil on the slide and switch to the oil
immersion objective
Change the objective to 100x oil immersion
Count 100 white cells including all cell lines from
immature to mature
Properly record and report results of differential cell
counts.
Note reference ranges and any abnormal results.

22. Evaluating Staining Properties
Locate a cell that can be positively identified,
such as a polysegmented neutrophil.
Note the staining characteristics of the nucleus
and cytoplasmic granules.
Relate these features to any unidentified cells
that may be present.

23. Evaluating Staining Properties
cont’d
Myelocytes and metamyelocytes, if present, are
recorded separately from neutrophils.
Band (stab) cells are generally counted as neutrophils
but it may be useful to record them separately.
An increase may point to an inflammatory process
even in the absence of an absolute leucocytosis.

24. Evaluating Staining Properties
cont’d
Remember that cells don’t always appear
“picture” perfect.
The actual staining color of cells may vary from
what is seen in textbooks or even in images.

25. Segmented Neutrophil
Approximately 40-75%
in peripheral blood
2-5 lobes of nucleus
Joined by thin filament
 Faint pink cytoplasm
sprinkled with pink or
purple neutrophilic granules

26. Small Lymphocyte
Approximately 40% in
peripheral smear
Small round nucleus,
clumpy, chunk
chromatin pattern
Notice the monocyte
on the top right

27. Large Lymphocyte
Large lymphocytes
are often confused
with monocytes
Remember - the
lymphocyte nuclear
chromatin is
compact, with
blocks of chromatin
The nuclear shape
may be round, oval
or indented

28. Monocyte
Approximately 5% in peripheral smear
Large, convoluted, brainy looking
nucleus with lacy looking chromatin
Pale, gray blue, ground glass
cytoplasm (numerous fine azurophilic
granules)
Monocyte nucleus can sometimes
take many shapes from a band
shape, to an oblong shape, to a
convolutes brainy shape.
What is important to remember is that
the chromatin is lace, open weaved,
and foamy looking.

29. Monocytes vs. Lymphocytes
Monocytes and
lymphocytes are often
confused
The chromatin pattern
of the monocyte is
light purple and
arranged in loose,
lacy or linear strands
The nucleus may be
round, lobulated or
folded

30. Basophil
Approximately 0-2% in peripheral
blood
Nucleus is bi-lobed and obscured
by granules
Cytoplasm is pale, washed out and
contains intense large blue black
granules
Granules are scattered throughout
the cell and it is hard to distinguish
any distinct nuclear characteristics.

31. Eosinophil
Approximately 2-7% in peripheral
blood
Bi-lobed or banded nucleus
Large, red-orange and distinct
granules
To identify the eosinophil rely on the
granules. The granules are very
individualized looking.
If the stain is right, then the cell will
look red orange.
If not, the cell will be washed-out
looking.

32. What Does a Normal Adult
Blood Smear Look Like?
Remember, percentages are approximate and
may differ with ethnic groups and in disease
conditions
What normal cell has not been discussed?
Segmented neutrophils ~60%
Lymphocytes ~30%
Monocytes ~5%
Eosinophils, Basophils ~3-5%

33. What About Bands?
This cell (also called a stab) is a
precursor cell to the segmented
neutrophil
The nucleus is C-shaped or horseshoe
shaped containing no filament and has
no tight or pinched constriction
Cytoplasm is pink gray with granules
Approximately 2-6% in normal
differential
If unsure of identification, classify
as a more mature cell, the
segmented neutrophil

34. What About Immature
Granulocytes?
Metamyelocytes
Myelocytes
Promyelocytes
Myeloblasts

35. Metamyelocyte
Also called a juvenile
Rarely seen normally in the peripheral blood
Nucleus characteristically indented or kidney-
shaped
The nuclear chromatin is clumped and
condensed
Specific granules appear the same as in the
band

36. Metamyelocyte (bottom) and Band (top)

37. Myelocyte
Should never be seen in the peripheral blood
The nucleus is round or oval and often
eccentrically located in the cell
The nuclear chromatin is beginning to clump; no
nucleoli are visible
A moderate amount of patchy blue cytoplasm
can be seen
The cell is characterized by the presence of light
pink or tan (neutrophilic) specific granules

39. Promyelocyte
Should never be seen in the peripheral blood
Large cell
The nucleus may be eccentric and slightly
indented with nucleoli still visible
The nuclear chromatin is loose and open
The cell is characterized by the presence of few
to many, prominent, dark red or purple
cytoplasmic granules

40. Promyelocyte

41. Myeloblast
Should never be seen in the peripheral blood
Large cell (size may vary)
The nucleus is large and round, with one or
more visible nucleoli
The nuclear chromatin is loose and open
The cytoplasm is scanty to moderate and deeply
basophilic

42. Myeloblast

43. Abnormal Inclusions in
Granulocytes (Neutrophils)
Toxic granulation
Dohle bodies
Vacuoles

44. Toxic Granulation
Large, blue-black or deep purple
granules in the cytoplasm of
neutrophils
Represent primary or
“azurophilic” granules
Seen associated with acute
infections, drug poisoning and
burns

45. Dohle Bodies
Small, round or oval light-blue
staining areas in the cytoplasm of
neutrophils
Represent aggregates of rough
endoplasmic reticulum
Seen in patients with infections,
burns and after exposure to
cytotoxic agents
May also be seen in conjunction with
toxic granulation

46. Vacuoles
Clear areas or “holes” in the
cytoplasm of neutrophils
Seen when the neutrophilic
cytoplasm begins to
degenerate or if the cell has
been actively phagocytic
Seen in patients with
infections and can be noted
along with toxic granulation
and Dohle bodies
N.B. Also can be seen as an
artifact associated with old
EDTA blood

47. Dohle
body
Toxic Granules
Vacuoles

48. Reactive Changes in
Lymphocytes
Response to a variety of viral and non-viral
stimuli produce diverse nuclear and/or
cytoplasmic changes in the lymphocytes
Associated with viral infections, e.g., Epstein-Barr
virus (EBV), cytomegalovirus (CMV), human
immunodeficiency virus (HIV)
Also seen in response to drugs or non-viral
organisms such as toxoplasmosis

49. Reactive Lymphocytes
Characteristics:
Irregular shape of cytoplasm and/or nucleus
Abundant dark blue cytoplasm, overall bigger cell
Cytoplasmic tags, sharp ridges; indented by red
cells
Increased number of reddish granules or vacuoles
in cytoplasm
Fine nuclear chromatin pattern; may see nucleoli

50. Reactive Lymphocytes

51. 8.3. Reporting the Differential White Cell
Count
The differential leukocyte count expressed as the
percentage of each type of cell
It should be related to the total leukocyte count
The results reported in absolute numbers as the
absolute value gives better clinical indication

52. Absolute vs. Relative Count
Absolute count
Derived by multiplying the
percentage of the identified
cell times the white count
If 40% lymphs are counted
and the white cell count is
5.0x10
9
/L then the absolute
lymphocyte count is
5.0x10
9
/L x.40 or 2.0x10
9
/L
This number is then
compared to the healthy
reference range
Relative count
Strictly the percent counted
in the WBC differential
The relative number is then
compared to the reference
range for normal white cell
differentials
So, it is possible for an
individual to have a relative
increase, an absolute
increase and a relative and
absolute increase of a
particular cell

53. Approximate Absolute and Relative Reference Ranges
Healthy Adult
Cell Type Absolute Relative
Segmented
Neutrophil
2.0-7.0 x 10
9
/L 40.0-75.0%
Lymphocyte 1.5-4.5 x 10
9
/L 20-45%
Monocyte 0.2-0.8 x10
9
/L 2-10%
Eosinophil 0.04-0.4 x 10
9
/L 0-7%
Basophil 0.02-0.1 x 10
9
/L 0-2%
Values may vary according to geographic location

54. Quality control
Well prepared well stained blood film which has
3 zones (Head, body, tail)
WBC should contain blue nucleus along with a
lighter staining cytoplasm
RBC should have good quality of color ranging
from buff pink to orange
Platelet should be blue with granules and no
nucleus

55. Sources of error
Use of unclean slide and improper smear
preparation and staining technique
Counting cells in an area not suitable for
counting
Misidentification of white cells
Interpersonal skill

56. 8.4. Interpretation of Differential White
cell count
For proper interpretation the relative count is of little use by
itself
For example, the fact that a sample may have 60%
polymorphs is of little use
A patient sample may have 60% neutrophil and a total
leukocyte count of 8.0 x 10
9
/L giving 4.8 x 10
9
/L neutrophils,
which is quite normal
but if the patient has 60% neutrophils in a total leucocyte count
of 3.0 x 10
9
/L, then the patient’s neutrophil count is1.8 x 10
9
/L
neutrophils. In this case the patient has granulocytopenia.

57. Interpretation cont’d
1.Neutrophil
1.1 Neutrophilia/neutrophilic leucocytosis:
an increase in the number of circulating neutrophils
above normal (>2.0-7.0 x 10
9
/L)
Overwhelming infections
Metabolic disorders: uremia, diabetic acidosis
Drugs and chemicals: lead, mercury, potassium
chlorate
Physical and emotional stress
Hematological disorders: myelogenous leukemia
Tissue destruction or necrosis: burns, surgical
operations

58. Interpretation cont’d
1.2.Neutropenia:
a reduction of the absolute neutrophil count below 2.0 x
10
9
/L
Myeloid hypoplasia
Drugs (chloramphenicol, phenylbutazone)
Ionizing radiation
1.3. Hypergranular neutrophils (neutrophils with toxic
granules): these are
neutrophils with coarse blue black or purple granules.
indicative of severe infection or other toxic conditions.

59. Interpretation cont’d..
1.4 Vacuolation
seen in progressive muscular dystrophy
1.5. Hypersegmentation:
neutrophils with more than six lobes to their nucleus
(as many as ten or twelve may be seen)
indicative of megaloblastic erythropoiesis (vitamin
B12 and/or folic acid deficiency), iron deficiency
anemia and uremia.
1.6. Agranular Neutrophils:
neutrophils devoid of granules
having a pale blue cytoplasm( features of leukemia)

60. Interpretation cont’d
2. Eosinophil
2.1. Eosinophilia:
an eosinophil count above 0.5 x 10
9
/L
Occurs during:

Allergic diseases: bronchial asthma, seasonal
rhinitis

Intestinal parasitic infections: e.g. trichinosis,
taeniasis

Skin disorders

Chronic myelogenous leukemia

61. Interpretation cont’d
2.2. Eosinopenia:
an eosinophil count below 0.04 x 10
9
/L
Occurs during:

Acute stress due to secretion of adrenal glucocorticoid
and epinephrine

Acute inflammatory states
3. Basophil
Basophilia:
a basophil count above 0.2 x 10
9
/L
Rare condition
Occurs during:
Allergic reactions
Chronic myelogenous leukemia
Polycythemia vera

62. Interpretation cont’d
4. Monocyte
4.1. Monocytosis:
a monocyte count above 1.0 x 109/l
Occurs during

Recovery from acute infections
Tuberculosis

Monocytic leukemia
4.2.Monocytopenia:
a monocyte count below 0.2 x 109/l
Occurs during

Treatment with prednisone

Hairy cell leukemia

63. Interpretation cont’d
5.Lymphocytes
5.1. Lymphocytosis:
absolute lymphocyte count above 4.0 x 10
9
/L in adults
and above 8.0 x 10
9
/L in children.
Seen during

Infectious lymphocytosis associated with coxackie
virus
Other viral infections: Epstein-Barr virus,
cytomegalovirus
Acute and chronic lymphocytic leukemia
Toxoplasmosis

64. Interpretation cont’d
5.2. Lymphocytopenia:
a lymphocyte count below 1.0 x 109/l in adults and
below 3.0 x 109/l in children
Seen in
Immune deficiency disorders: HIV/AIDS
Drugs, radiation therapy
Atypical lymphocytes:
Large cell; abundant pale blue cytoplasm with
peripheral basophilia, may have azurophilic granules
They are primarily seen in
infectious mononucleosis which is an acute, self-
limiting infectious disease of the reticuloendothelial
tissues, especially the lymphatic tissues

65. Summary: Performing
Differential Cell Counts
Use properly prepared and stained blood smears
Establish a good counting area
Properly identify white cells
Provide an estimate of total white cells and platelets
Record cellular morphology (RBC, WBC, and Plt)
Properly record and report results noting reference
ranges and abnormal results

66. Review Questions
1.Define differential leukocyte count.
2.What is the importance reporting the differential
leukocyte counts in absolute terms?
3.What other elements of the blood film should be
evaluated while doing the differential leukocyte count?
4.Describe three tracking methods of diff counting with
their advantage and disadvantage
5.List at least five factors affecting the differential white
cell count and their remedies