Leukocytes: The Granulocytic and

Monocytic Series
Part II

Chapter 14
The Leukocytes (part 2)
(monocytes and lymphocytes)
Learning objectives:
Upon completion of this lecture series the learner should be
able to competently discuss:
• The differentiation and maturation of the granulocyte cell
series in detail
• The differentiation and maturation of the monocytemacrophage cell series in detail
• The function of granulocytes and monocytes
•Methods of assessment of granulocytes and monocytes in a
clinical setting (absolute counts, differential counts, morphology,
staining patterns, etc.)

Monocyte-macrophage cell series
Types of Macrophages: the name depending of the tissue
where they are located
1.

Histiocytes in connective tissue

2.

Kupffer Cells in liver

3.

Osteoclasts in bone

4.

Microglial in nervous system

 These cells plus the reticular cells of primary and secondary
lymphoid organs (thymus, spleen, etc.)  mononuclear
phagocytes (Mononuclear cells are distributed throughout the
body)
 PMNs as the major phagocytes (segmented neutrophils) remain in
circulation unless recruited to sites of injury / infection.

Maturation of monocytes
• Lineage: CFU-GM  CFU-M  monoblast  promonocyte  monocyte

2-3 mitotic
divisions in 2 days
(to bring number of differentiating cells up)
• This developmental pathway in BM occurs under the influence of several
growth factors; chief among them is the M-CSF
• Monocytes are mostly released into circulation within 12-14 hrs after
maturation in BM  No large reserve of cells in BM maturation/storing
compartment as do the granulocytes
• In circulation, monocytes locate into circulation & marginating pool @ a
ratio of 1:3.5

Maturation of monocytes
Monoblast:
(morphologically indistinguishable

from myeloblast by light microscopy
Except for highly experienced hematologist )

Size: 14 to 20 µm
N/C ratio: High
Nucleus: Oval, round, or folded
Chromatin: finely dispersed not clumped
(lacy-like), light blue purple
Nucleoli: several
Cytoplasm: agranular, blue gray.

Promonocyte
Size: 12 to 20 µm

N/C ratio: Moderate (N ↓in size)
Nucleus: Irregular and folded
Chromatin: Coarser than
monoblast
Nucleoli: May be present
Cytoplasm: abundant, blue gray.
Granules: Azurophilic may

present

Monocyte
Size: 12 to 20 µm (Largest mature cells in
the periphery)
Nucleus: Folded, horseshoe,
bean-shaped
Chromatin: Loose and linear,
lacy pattern when compared with
granulocytes
Nucleoli: rarely are present
Cytoplasm: blue gray, ground glass
appearance, ; exhibit irregular outline showing
pseudopodes and vacuoles
Granules: Fine, dust-like and membrane-bound granules

Monoblast: Moderate amount of gray-blue cytoplasm. The
centrally located nucleus is round with a fine chromatin
pattern.

Promonocyte: Abundant gray blue cytoplasm with mildly irregular outline & few
granules, minor vaculation, nucleus is folded and clumped.

Monocyte: Abundant vacuolated gray blue cytoplasm with irregular outline and few
granules. Nucleus is kidney bean–shaped and clumped.

Summary of granulocyte & monocyte functions
Granulocytes and monocytes represent a significant part of the
innate arm of the immune system  participate in various
immune mechanisms
Eosinophils
– Primary function is against helminthes (parasites).
– Eosinophilia may occur in allergic conditions and with
parasitic infections, and with chronic inflammation.
Basophils:
– Mediators of the inflammatory response, particularly in
hypersensitivity reactions  Release of inflammatory
mediators (histamine) during allergic reactions is mediated
by basophils and mast cells (in the presence of IgE)

Mature neutrophils (phagocytes)
Express cell surface markers that correlate with their functional state:
– Receptors for adhesion: CD11b, CD62L, CD49d, CD54
– IgG receptors {FcγRI (CD64), FcγRIIA (CD32)and FcγRIII(CD16) }
– CD32 (down-regulates IgG production by B cells)
– Anaphylatoxin (C3a, C4a and C5a) receptors eg CD88(C5a
peptide)
– TLRs
Macrophages (phagocytes)
Variably express CD14, CD16, CD45, IgG receptors, TLRs, etc. 
different functional subsets. Example:
– CD14+CD16- = classical monocytes  excellent
phagocytes but poor producers of inflammatory
cytokines
– CD14lowCD16+ = nonclassical monocytes  good APCs

Summary of granulocyte & monocyte functions (cont.)
 Neutrophils and macrophages carry out phagocytosis as well
as antigen processing and presentation (so as to engage
adaptive immunity)

 Dendritic cells (DCs), which arise from monocytes and
express multiple TLRs and T cell co-stimulatory molecules
like CD80/CD86, CD40 etc., are excellent APCs especially to
Th cells.
 Release of inflammatory mediators (cytokines): neutrophils,
monocytes/macrophages, & DCs

The acute inflammatory response
• Injury/ infection  pathogen-related antigens (e.g. LPS,
proteins, etc.) trigger complement activation and release
of inflammatory mediators (cytokines like TNF- & IL1, IL-8, etc.)
• Activation of complement 
– release of C3b (coats pathogen to initiate MAC
formation and/or to facilitate phagocytosis)
– release of chemo-attractants like C5b.
• C3b, C4a and C5b trigger degranulation of mast cells
and release of histamine which along with TF and other
mediators induce muscle contraction and vasodilatation

 Pathogen-related antigens plus mediators up-regulate
expression of homing markers like sialyl-Lewisx on
neutrophils on in circulation.
• P-selectin+ (CD62P+) on vascular endothelium cells
recognize and bind sialyl-Lewisx neutrophils arrest at
site of infection.
• Neutrophils up-regulate expression of certain integrins
(e.g. CD11b) on their surface and bind with VCAM,
ICAM on vascular endothelium cells  further adhere to
vascular endothelium and
• Neutrophils exit circulation by diapedesis through PCAM on endothelial cells and neutrophils.
• On site, neutrophils start phagocytosing (eliminating)
C3b-coated pathogens.

Phagocytosis

Digestion:
• 1)The action of the oxygen-dependent myeloperoxidase-mediated system,
hydrogen peroxide, and an oxidizable cofactor serve as major factors in the actual
killing of bacteria within the vacuole.

• 2) Other oxygen-independent systems, such as alterations in pH, lysozymes,
lactoferrin, and the granular cationic proteins, also participate in the bactericidal
process.

Antigen processing and presentation

Methods of Assessment for
leukocytes

• Normally only mature cells are released into the peripheral blood.
Mature cells may also remain stored in BM.
• Increase / decrease in WBC count may be caused by an alteration
of all WBC cell lines, but more commonly results from an
alteration of only one type of WBC  Hence, a differential count
is important.
• From the differential and the WBC count, the absolute values for
each type of WBC can be calculated (relative differential (%) x
total WBC count).
• Most variations in the leukocyte count are due to increases or
decreases in the number of neutrophils since, by percentage,
they are the most numerous.

Reference ranges of granulocytes and monocytes

Total number and range vary with age, gender, and race  consult
appropriate ref. values for interpretation of results.

Examples of WBC value shifting:
• Blacks have lower total leukocyte count as average neutrophil
counts are lower than in whites
• Smoking slightly elevates percentage of neutrophils
• Eosinophils exhibit hormone-related daily fluctuations
(diurnal) (high at night\sleeping time).
• Count and distribution also varies with health status of the
host; most significant in clinical practice.

Generally, leukocyte concentration in blood is as follows :
1. Total WBC count
• At birth: 9-30 X 109/L
• In children (average): 8 X 109/L
• In adults: 3.5-11.0 X 109/L
2. Differential count (adults)
• Segmented neutrophils: 40-75%
• Neutrophils-bands: 0-3%
• Eosinophils: 1-6%
• Basophils: 0.5-1% (rarest type)
• Lymphocytes: 25-40% (up to 60% in children)
• Monocytes: 2-8%

Fluctuations in total Leukocyte Count
Increases:

Acute inflammation

Pregnancy

Strenuous exercise
Decreases:

Sepsis (infection-related systemic inflammation)  causes
significant tissue damage)

Immunosuppressive agents (steroids, antineoplastic agents, HRT,
etc)
I. Absolute Cell Value =
Total Leukocyte count X Percentage of Cell Type
Example: Patient Data
Total Leukocyte Count = 15.0 X 109/L
Differential blood smear results: Bands: 12%, segmented neutrophils 80%,
lymphocytes 8%?

II. Differential Blood Smear Evaluation
• Observe abnormal cells
• Observe percentage of each type of
leukocytes
• Observe presence of band forms; normal
range of band forms in adults: 0-3%
• Observe Shift to the left: increased band forms
with the presence of metamyelocyte and
myelocytes.

Shift to Left in granulocytes = increased percentage of immature/total
leukocytes in cases of infection , inflammation, cancers and certain types
of leukemia, etc.

III. Assessment of Eosinophils and Basophiles
When % of eosinophils increase, absolute count is recommended
because:
– Eosinophil count rises during parasitic worm infections
(roundworm, hookworm), rheumatoid arthritis, inflammatory
bowel disease, autoimmune diseases, ….
– Eosinophils may increase due to Hodgkin’s lymphoma, lung
and stomach cancer; any neoplams (cancer) or leukemias
can increase the eosinophil count.
– Allergies (hay fever, asthma, ..) can increase eosinophils.
Basophiles are the least numerous of granulocytes in circulation.
Normally, 1-2 basophiles are seen on differential. Increased % is
seen in conditions such as chronic myelogenous leukemia and
polycythemia vera.

IV. Leukocyte Alkaline Phosphatase Test:
This special test, which measures the amount of the ALP

in test cells, is performed to differentiate malignant
disorders [e.g. chronic granulocytic leukemia] from the

leukemoid reaction [leukocytosis exceeding 50,000
WBC/mm3 with a significant increase in early

neutrophil precursors] as seen in severe infections.
* High readings = leukomoid reaction

LAP Test
• LAP is found within white blood cells, WBC levels of
LAP can help in the diagnosis of certain conditions.
– Higher levels: are seen in polycythemia vera (PV),

essential thrombocytosis (ET), primary myelofibrosis
(PM), and the leukemoid reaction.

– Lower levels : are found in chronic myelogenous
leukemia (CML), paroxysmal nocturnal
hemoglobinuria (PNH) and acute myelogenous
leukaemia (AML).

Neutrophilic Hypersegmentation
Index
• Mature segmented neutrophils have from two to five nuclear
lobes (segments).
• Counting the number of lobes can be performed to determine
the neutrophilic hypersegmentation index (NHI).
• The NHI is clinically useful in vitamin B12 deficiency (pernicious
anemia) and folic acid diagnosis.

1. Lobe average: This is determined by counting the number
of lobes in a number of neutrophils. The reference value is 2.5 to 3.3.
2. Percentage of neutrophils with five or more lobes to the total
neutrophils.
3. Hypersegmentation index. To calculate this index,
Number of neutrophils with 5 or more lobes × 100
Number of neutrophils with 4 lobes
Values greater than 16.9 are considered to indicate
hypersegmentation.
This method is considered to be the most sensitive method.

WBCs
Histogram

WBCs Histogram
3-Part Differentiation

LYSE Reagent effect on WBCs WBCs Histogram
Before adding Lyse Reagent

3-Part Differentiation

Cell diameter
in µm

Neutrophils 10 – 15
Basophils 9 – 14
Eosinophils 11 – 16
Monocytes 12 – 20
Lymphocytes7 – 12
0

2 4 6 8 10 12 14 16 18 20 22 µm

LYSE Reagent effect on WBCs

WBCs Histogram
3-Part Differentiation

After

adding Lyse Reagent

• Lyse reagent causes
WBCs to shrink to a
defined
volume
according to their
cell type.

Lymph

• WBCs are presented as
3 separate populations
in the histogram.

100

200

300 fL

LYSE Reagent effect on WBCs WBCs Histogram
After

adding Lyse Reagent

3-Part Differentiation

• Lyse reagent causes
WBCs to shrink to a
defined
volume
according to their
cell type.

Lymph
Mono

• WBCs are presented as
3 separate populations
in the histogram.

Eosino
Baso

100

200

300 fL

LYSE Reagent effect on WBCs WBCs Histogram
3-Part Differentiation

After

adding Lyse Reagent

Neutrophils

Lymph

• WBCs are presented as
3 separate populations
in the histogram.

Mono

Eosino
Baso

100

• Lyse reagent causes
WBCs to shrink to a
defined
volume
according to their
cell type.

200

300 fL

LYSE Reagent effect on WBCs WBCs Histogram
After

adding Lyse Reagent

3-Part Differentiation

• Cells will be shown in a histogram according to their size.
Lymphocytes30 – 80 fL

Neutrophils

Lymph

Monocytes 60 – 120fL

Mono

Eosino

Basophils 70 – 130fL

Baso

Eosinophils 80 – 140fL
100

200

300 fL

fL
Neutrophils120 – 250

WBCs counted between
2 outer
discriminators

WBCs Histogram
3-Part Differentiation

WL WBCs Lower discriminator
= LD Lower Discriminator

WL
LD

[ Flexible ] fluctuates between 30 – 60 fL

%

100

200

300 fL

WBCs counted between

WBCs Histogram

2 outer
discriminators

3-Part Differentiation

WL

WU
UD

LD
%

WU WBCs Upper discriminator
= UD Upper Discriminator
[ Fixed ] at 300 fL

100

200

300 fL

WBCs histogram consists of 2
Troughs
[ Valleys ]

WBCs Histogram
3-Part Differentiation

[ Detected by 2 inner discriminators]
WL
LD

WU
UD

T1 T2

%

T1 : 78 – 114 fL
T2 : > 150 fL

100

200

300 fL

WBCs Histogram

WBCs histogram consists of 2
Troughs
[ Valleys ]

3-Part Differentiation

[ Detected by 2 inner discriminators]
WL
LD

WU
UD

T1 T2

%

Peak between LD and T1
represents small cells
i.e. Lymphocytes

100

200

300 fL

WBCs histogram consists of 2
Troughs
[ Valleys ]

WBCs Histogram
3-Part Differentiation

[ Detected by 2 inner discriminators]

Peak between T1 and T2 includes:

WL
LD

WU
UD

T1 T2

%

100

200

300 fL







Eosinophils
Monocytes
Basophils
Blast cells
Promyelocytes
Myelocytes
Metamyelo
cytes

WBCs histogram consists of 2
Troughs
[ Valleys ]

WBCs Histogram
3-Part Differentiation

[ Detected by 2 inner discriminators]
WL
LD

WU
UD

T1 T2

%

Peak after T2 represen
Neutrophils

100

200

300 fL

WBCs Histogram

Important Notes
WL
LD

WU
UD

T1 T2

%

100

3-Part Differentiation

200

The distribution curve
should be within the
discriminators, starts and
ends at the base line.

Important Notes
WL
LD

T1 T2

%
30 – 60 fL

100

200

WBCs Histogram
3-Part Differentiation
The distribution curve should
be within the discriminators,
starts and ends at the base
line.
WU The LD is flexible while UD is
UD fixed.
At 300 fL The WBC-channel shows only
WBCs and platelets (RBCs are
lysed)
The volume of the platelet is
usually between 8 - 12 fl,
therefore the LD at the WBC
Histogram seperates the
WBCs
from
the
platelets.(Platelets were not
counted).