White Blood Cells

&
Benign Disorders
By
Prof. Afaf Abdel Aziz
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 White Blood Cells
1- Development:
Site Of Haemopoiesis:
Fetus:
0-2 months yolk sac.
2-7 months liver, spleen .
5-9 months BM .

Infants BM ( Practically all bones ).

Adults BM of vertebrae, ribs , sternum, skull,
sacrum, pelvis & proximal end of femur.

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Haemopoiesis:

3
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Control of Haemopoiesis

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Haemopoietic growth factors:
• Glycoprotein hormones that regulate
proliferation, differentiation of progenitors
&function of mature cells.
• The effect is mediated through specific
receptors on target cells.
• Act locally at site of production or circulate
in plasma.

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Kinetics of WBCs
•N BM: 6-10 day
PB: 6-10 hours
Tissues: 4-5 days.
In BM:
Mitotic pool (myeloblast, promyelocyte, myelocyte)
Postmitotic:
Maturation metamyelocyte, band, segmented
Storage pool (reserve pool) band, segmented
(which is affected by steroid, inflammation, infection).

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Blood Circulating granulocyte pool (blood count)
epinephrine, acute stress & exercises

Marginated

Tissues die in subclinical infection

senescence
migrate out in body secretion
(urine, saliva, gut)

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E BM: 3-6days to mature.
PB: 30 minute
Tissues:12 days

B PB: 2-5 days

Tissues: mast cells

M PB: 1-3 days

Tissues: main site of action, differentiate
into macrophage

L T-lymphocyte: can be very long lived

B-lymphocyte:4 or more weeks.
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Functions of WBCs
N
1- Major front line defense against infection d.t their ability to
ingest & kill micro-organisms.
2- Immunomodulatory role because mature cells can secrete a
variety of cytokines in response to stimulation.

E
1- Less phagocytic & bactericidal activity than N
2- Important role in mediating hypersensitivity reactions,
bronchial asthma & skin inflammation.
3- Involved in chronic inflammatory response.
4- Provide protection against parasites aided by T- helper
lymphocytes.

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 B Both B& mast cells have a major role in hypersensitivity
diseases.

M
1. Scavenge debris, aid wound healing, sequester & detoxify
injurious material, remove senescent or dying cells.
2. Bone resorption & remodeling.
3. Mediate killing of tumor cells.
4. Destroy invading pathogen.
5. Major role in the control of 1ry & 2ry immune response.
6. Regulate other cells involved in the immune response such as T &
B lymphocytes by antigen presentation & cytokine secretion.
7. Contribute to regulation of hemopoiesis, hemostasis &
fibrinolysis.
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Normal Values in Adults:
9
• TLC: 7.0 + 3.0 x 10 /L
Absolute Relative
9
• Neutrophil (N) 2.0- 7.0x10 /L (40-80%)
9
• Lymphocytes (L) 1.0- 3.01x0 /L (20-40%)
9
• Monocytes (M) 0.2-1.0x10 /L (2-10%)
9
• Eosinophils (E) 0.02-0.5x10 /L (1-6%)
9
• Basophils (B) 0.02-0.1x10 /L (0-2%)

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Infants & children:
X109/L Full term Day 3 1 month 2-6 3-6 6-12
months years years

TLC 18+8 15+8 12+7 12+6 10+5 9+4

N 5-13 3-5 3-9 1.5- 1.5-8 2-8

9

L 3-10 2-8 3-16 4-10 6-9 1-5

M 0.7-1.5 0.5-1 0.3-1 0.1-1 0.1-1 0.1-1

E 0.2-1 0.1-2.5 0.2-1 0.2-1 0.2-1 0.1-1

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• Absolute value:
Relative value x TLC
100

• Granulocyte variations:
1- Numerical variation: increase or decrease.
2- Morphological variation:

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Leucocytosis: increase in the number of circulating leucocytes, above
normal range.

-Neutrophilia -Eosinophilia -Lymphocytosis

-Basophilia -Monocytosis
-Leukemoid reaction -Leucoerythroblastic reaction

Leucopenia: Reduction in the number of leucocytes

• It may affect one or more lineages.
• It is possible for a case to be severely neutropenic or
lymphopenic without a reduction in the total WBCs count.
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Causes of Neutrophil Leucocytosis (Neutrophilia):
1- Bacterial infections (esp. pyogenic bacterial, localized or
generalized).
2- Inflammation & tissue necrosis (e.g., myositis, vasculititis,
cardiac infarction, trauma).
3- Metabolic disorders (e.g., uremia, eclampsia, acidosis,
gout).
4- Neoplasms of all types.
5- Acute hemorrhage or haemolyosis.
6- Corticosteroid therapy (inhibits migration).
7- Myeloproliferative disorders.
8- Treatment with myeloid growth factors (GM-CSF & G-CSF).
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The cause is obvious form a careful history & examination

Reactive neutrophilia may be associated with shift to left

(increase in the number of immature forms), toxic granulations
in the cytoplasm, Dohle bodies & high neutrophil Alk
Phosphatase score (N: 20-100).

If the count is very high = leukemoid reaction.

NB: If no immediate cause can be found, the possibility of CML

or other MPDs.

Physiologic neutrophilia is caused by vigorous exercise, after

the injection of epinephrine, during pregnancy.
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Causes of eosinophilia:
1- Allergic diseases.
2- Parasitic infestation.
3- Certain skin diseases e.g psoriasis, pemphigus & dermatitis
herpetiformis.
4- Pulmonary eosinophilia & hypereosinophilic syndrome.
5- Drug sensitivity.
6- Polyarteritis nodosa.
7- Hodgkin‘s disease.
8- Eosinophilic leukemia.
9- Treatment with GM-CSF.
10- Recovery from acute infection.
11- Following irradiation or splenectomy.
12- Drugs.
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 If you find eosinophilia, you should search for a cause.
If no primary cause + complex symptoms HES.

Persistent elevation of E ( > 1.5 x 109/L) for 6 month (no cause)

Organ damage, heart, lung (cough, dyspnea), skin (pruritis).
Middle aged males.
Fever & weight loss.
Untreated: median survival 1 year.
Treatment: corticosteroids, cytotoxic agents (hydroxyurea), IFN-
alpha, cyclosporine A.
N B: long term follow up is essential to ensure that patient does
not have lymphoma or MPDs.
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Causes of lymphocytosis:
1-Infections:

Acute: infectious mononucleosis, rubella, mumps, pertusis, acute

infectious lymphocytosis, infective hepatitis, herpes.
Chronic: T.B, syphilis, brucella, Toxoplasmosis.
2- CLL, PLL.
3- ALL.
4- Non-Hodgkin’s lymphoma.
5- Hairy cell leukemia.
Acute infectious lymphocytosis:
- occurs in children & in small epidemics.
- the cause may be enterovirus.
- the count reaches 25-100 x 109/L, morphologically normal
lymphocytes.
Pertusis: extreme degree of lymphocytosis up to 150 x 109/L.
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Causes of Monocytosis:
1- Chronic bacterial infections: T.B, brucellosis, bacterial endocarditis,
typhoid.
- Recovery from acute infections.
2- Protozoon infections.
3- Hodgkin’s disease.
4- CMML.
5- Acute monocytic leukemia & myelomonocytic leuk.
6- Treatment with GM-CSF or M-CSF.
7- Chronic neutropenia

Note: In the developing world, an apparently unexplained

persistent monocytosis is most likely to represent an occult
chronic infection, but in the developed world, the most
common cause is CMML.
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Causes of Basophilia:
1. Chicken pox, chronic ulcerative colitis.
2. Myxoedema.
3. Myeloproliferative disorders esp. PR, CML &
Increased in transformation to blastic phase of CML.
4. Inflammatory conditions.

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Leucoerythoblastic Reaction:
Appearance of granulocyte precursors & nucleated red cells in the
P.B.
 TLC is N or moderately increased.
Causes:
Metastatic carcinoma in the marrow.
Myelosclerosis.
Multiple myeloma.
,
Hodgkin s disease.
Miscellaneous conditions:
 Miliary T.B.
 Thrombotic thrombocytopenic purpura.
 Megaloblastic anemia.
 Severe hemolytic anemia.

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Leukmoid Reactions:
- Extremely high leukocyte counts.
- Non-leukemic conditions.
- TLC > 50 x 109/L .
- Granulocytic or lymphocytic.
- Causes:
• Severe infections esp. in children: pneumonia, septicemia,
meningococcal meningitis, T.B, Inf. Mononucleosis, inf.
Lymphocytosis, pertusis.
• Intoxications: eclampsia, severe burns.
• Neoplasia: esp. with bone marrow metastases.
• Severe hemorrhage or hemolysis.

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Other varieties of leukaemoid reactions (rare):
Monocytic leukemoid reaction:
Reported in a case associated with T.B involving lymph nodes,
lung, liver & coexisting mediastinal teratoma.
The leucocytes count reached 82 x 109/L with 42% monocytes.

Eosinophilic leukemoid reaction:

Reported in association with amebiasis, lung cancer &
melanomatosis.
Very high concentration of eosinophils.

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 Comparison of leukemoid Reaction & Leukemia

Leukemoid Reaction CML

Clinical Picture features of causative disorder is obvious Splenomegaly, L.N enlargement ??

TLC - moderate , rarely exceed 100 x 109/L often exceed 100 x 109/L
No. of immature cells - usually mild or moderate Numerous
- myelocytes rarely exceed 5-15%, blasts 1-5%
Morphology - toxic granules in infective cases toxic changes uncommon, hypogran. & Pelger Huet

Anemia varies with cause, but often mild or absent usually marked & progressive

Nucleated RBCs - frequent in leuco-erythroblastic reaction Infrequent

Platelets N or or in leuco-erythoblastic reaction , N, according to stage

B.M Mild myeloid hyperplasia Marked hyperplasia

NAP.score High score Low score

Ph+ chromosome -ve +ve

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• Destruction by circulating antibodies
• Direct BM suppression by toxins
• Depleation of marrow stores by release into the
circulation and migration to the site of infection

Viruses produce sever dangerous neutropenia

eg. HBV,HIV-1

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Mechanism:(cont.)
- Direct viral nfection and damage to HSC
-Antineutrophil antibodies production

B- Drugs and chemicals:

Either produce - Pancytopenia:
eg. Chemotherapy

-Neutropenia & agranulocytosis

eg. Analgesics
Antimicrobials , Anticonvulsants , Antihistaminics
Antimalarials ,
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Drugs& chemicals(cont.)
Mechanism :
* Immune destruction of granulocytes and its precursors
*Dose dependent inhibition of granulopoiesis
*Direct toxic effect on myelopoiesis or marrow
microenvironment

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 TLC

C. Nutritional: N

• Cachexia & debilitated states (starvation, anorexia

nervosa, marasmus)mechanism impaired blood cell
production caused by lack of protein building blocks
• B12 & folate deficiency mechanism interfere with nucleic
acid synthesis of myeloid precursors
• Copper deficiency

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D. Immune neutropenia:
• Isoimmune neonatal neutropenia [like Rh]
• Chronic auto-immune neutropenia [like autoimmune
hemolytic anemia or thrombocytopenia]
• T- γ lymphocytosis: auto-immune neutropenia (±
thrombocytopenia or anemia) + BM infiltration with
large granular lymphocytes
• Following BM transplantation
• Reaction to transfused blood products

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Mechanism of immune neutropenia
Mediated by specific antineutrophil
antibodies destruction of N by splenic
sequestrates of opsonised cells or by
complement mediated lysis

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E. Felty’s syndrome:
Triad of:
• Rhoid arthritis
• Splenomegaly
• Neutropenia
Mechanism :
Like immune neutropenia + splenomegaly

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F. Neutopenia associated with
complement activation
• Dialysis, bypass
• Anaphylactoid shock
• Apheresis

G. Splenic sequestration:
In all cases with splenomegaly
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 3. Congenital or chronic neutropenia:

a) Severe congenital neutropenia (Kostmann syndrome):

• Severe chronic neutropenia .. Frequent pyogenic infections
• In most patients the E & M are normal or high
• The BM is usually hypercellular with “maturation arrest” at or
beyond the promyelocyte stage
• Very poor prognosis, but most patients respond well to daily G-
CSF injections

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• Few children have developed leukemia while
on G-CSF
• An error in initial diagnosis
• An effect of G-CSF administration
• Natural history of the disease masked previously
by early death from sepsis
• It may be due to defect in G-CSF signaling
pathway

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b) Cyclic neutropenia:
• An unusual disorder
• Familial, autosomal dominant
• Characterized by regularly recurring
neutopenic episodes occurring every 3 weeks,
lasts for 3-6 days and may be severe
• Associated with oral ulceration & infection
• Cyclic neutropenia is stem cell disorder due to
impaired response of marrow progenitor cells
to growth factor stimulation
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 c) Chronic benign neutropenia:

Familial Non-familial
• Benign • Benign
• Border line low N count • Severe decrease in N count
• Asymptomatic • Patients don’t suffer from
frequent infections
Or
• Infections if present: skin &
oral cavity infection
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 d) Idiopathic chronic severe
neutropenia
• Chronic symptomatic, severe
• Later childhood or during adult-hood
• No underlying etiology

e) Neutropenias associated with

congenital immune defects
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4. Part of general pancytopenia
• Aplastic anemia
• BM infiltration
• Leukemia
• Hypersplenism
• MM
• MDs
• PNH
• MF
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Lymphopenia
I. Primary: primary immune defiecency
II. Secondary:
• Infections: influenza; occasionally: other viral infections, miliary
T.B, pneumonia, septicaemia, malaria, HIV
• Loss of lymphocytes:
– Intestinal lymphangiectasia
– Lymphatic fistula

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• Therapeutic procedures:
– Radiotherapy
– Corticosteroid
– Anti lymphocyte globulin
– Cytotoxic drugs
• Neoplastic conditions:
• Metastatic carcinoma
• Advanced hodgkin’s disease

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• Nutritional metabolic:
– B12 or folate deficiency
– Zinc deficiency
– Uraemia
• Other conditions:
• SLE
• Other collagen vascular diseases
• Myasthenia gravis, aplastic anaemia
• Graft versus host disease, idipathic

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 Mechanism of Lymphopenia
• Some lymphopenias are due to redistribution
rather than depletion of total body lymphocyte
number
• Retention of lymphocytes in secondary
lymphoid organs caused by:
1. Corticosteroid therapy.. Dramatic short term
lymphopenia which become normal after two days
2. Endogenous secretion of corticosteroids during
acute illness e.g heart failure, pneumonia
• Increase rate of death of lymphocytes and/or
reduction of their rate of formation
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Eosinophilopenia
1. Acute infection & inflammation
2. Acute inflammation in patients with parasitic induced
eosinophilia due to:
• Abrupt decrease in circulating eosinophils:
– Intravascular migration
– Intravascular destruction and/or migration into tissues
• Inhibition of eosinophil release from the marrow
• Delayed suppression of eosinopoiesis

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Infectious Mononucleosis
Cause:
• Epstein-Barr virus (EBV)
• Infects certain epithelial cells & B lymphocytes
• Virus enters B cells via CD21 (surface receptor for the C3d
component of complement)

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Clinical picture:
• Incubation period: 7 weeks
• Fever: mild --- severe
• Sore throat: in > 50% of cases
• Lymphadenopathy:
• Bilateral cervical in 75%
• Generalized in 50% ± fever
• Palpable spleen: > 50%
• Hepatomegaly: ≈ 15%
• Jaundice: 5%
• Morbilliform rash
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Blood picture:
• TLC:
 10 – 20 x 109/L in ⅔ of cases
 Some patients have higher counts
 It peaks on 2nd & 3rd week, persists for 1-3 months
• Diff:
– Absolute lymphocytosis with normal small
lymphocytes + large numbers of
• Atypical lymphocytes
• Activated T cells
• Variable in appearance [their greatest number is usually
found between 7th & 10th days of illness
– Neutropenia
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Atypical lymphocytes

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• Anaemia:
 Rare, auto-immune haemolytic anaemia
 IgM of cold type anti-i-antibodies
• Thrombocytopenia:
 May occur & is occasionally severe
 Auto-immune
Three categories of antibody are produced as a
result of EBV infection:
1. Virus specific
2. Heterophile
3. Auto-immune

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Virus specific antibodies:
1. Immunoglobulin M anti-viral capsid antigen
(VCA) antibodies
• Develop during the IP & they peak in the second week of
the illness, followed by rapid decline
2. IgG anti VCA antibodies peak in the 2nd to 3rd
week & persists for life
3. Most patients have a transient response to EB
early antigen (EA). It peaks in 2nd & 3rd week
4. Antibodies to EBV nuclear antigens(EBNA-1 &
EBNA-2): develop later and last for life

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N.B serelogical diagnosis of the acute IM is most
accurately made by the presence of:
IgM anti-VCA & anti-EA antibodies + IgG anti-VCA
& absence of: anti-EBNA antibodies
“IgG anti-VCA + anti-EBNA + absence of IgM anti-VCA =
past infection”

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Heterophil antibodies:
• Serum agglutinins against sheep RBCs, persists for 6
weeks
• Paul-Bunnell test: titre > 1:112 is highly suggestive of IM
(absorbed by ox RBCs)
• Slide monospot test (using formaline treated horse RBCs)
• Similar antibodies are found in low titre in healthy
individuals & in some leukaemias & lymphomas & serum
sickness (absorbed out on guinca-pig red cells & kidney)
• When heterophils are negative EBV- specific serodiagostic
tests should be applied

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Auto-immune antibodies:
• Cold-reactive anti-i-antibodies
• Occasionally: anti smooth muscle, thyroid, stomach,
rhoid factor, antinuclear
Differential diagnosis:
• Acute leukemia
• Cytomegalovirus infection
• Toxoplasmosis
• Influenza, rubella, infectious hepatitis
• Bacterial tonsillitis

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Neutrophil granules:
• Mature neutophils contain
1. Primary granules: (azurophil granules):
myeloperoxidase appear at promyelo..reduced at
myelocyte stage
2. Secondary (specific granules):
• Produced during myelocyte stage
• Give the cytoplasm its pink background colour
Lysozymes, collagenase, vit. B12-binding protein, lactoferrin

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3. Tertiary granules:
Gelatinase
4. Phosphasomes:
Very light granules alkaline phosphatase compared
to specific granules
5. Secretory granules:
Plasma protein-complement , tyrosine kinase

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Neutrophil function
1. Chemotaxis & motility
2. Opsonization
3. Phagocytosis, degranulation, O2 radical release
4. Intracellular killing

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Morphological variations
Congenital
Barr bodies:
A ‘Drum stick’ appendage is present on the nucleus of a
proprotein of N from normal female due to the presence of
two x chromosomes

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Pelger-Huet anomaly:
• Uncommon autosomal recessive disorder
• Nucleus is bi-lobed and occasionally unsegmented
Neutrophil hypersegmentation:
• Rare autosomal dominant condition
• 5 lobes or more

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May-hegglin anomaly:
• Rare autosomal dominant
• N contains basophilic inclusions of RNA
• More frequently associated with mild
thrombocytopenia & giant platelets
• Occasionally associated with neutropenia

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Alders’s anomaly:
• Rare autosomal recessive
• Granulocytes, lymphcytes & monocytes
contain granules which stain purple with
Romanowsky stains
• These granules contain mucopolysaccharides

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Chediak-Higashi syndrome:
• Autosomal recessive
• Giant granules in granulocytes, monocytes &
lymphocytes + leucopenia + impaired function

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Acquired
Toxic granulations: in infection
Hyper segmentations:
• Megaloplastic anameia
• Renal failure
• Cytotoxic drugs esp. methotrexate & hydroxyurea
Pseudopelger:
• Myelodysplasia
• AML

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Functional disorders of Leucocytes
Congenital
Myeloperioxidase deficiency (MPO):
• Inherited MPO deficiency
• The most common disorder of neutrophil function
• No increased frequency of infection due to the presence of
MPO-independent microbicidal mechanisms

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 Chronic granulomatous disease of
childhood (CGD):
• Rare clinical syndrome
• Inherited defects of leucocyte functions
• Defective respiratory burst during phagocytosis & inability to
generate oxygen radicals in neutrophils, eosinophils,
monocytes & macrophage
• Recurrent bacterial & fungal infections esp. skin & lung
which are life threatening
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Leucocyte adhesion deficiency
• Rare autosomal recessive disorder
• Life threatening
• Profound deficiency or complete lack of leucocyte integrin
expression (β2 integrin chain)
• Recurrent indotent & necrotic soft tissue infection
involving mainly skin, mucous membranes &
gastrointestinal tract
• Bacterial infections predominate but fungal & viral
infections also occur
• Defect in adherence/movement/migration
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Neutrophil-specific granule deficiency
• Very rare autosomal recessive disorder
• Deficiency of neutrophil-specific granules, lactoferrin,
transcobalamin I & cytochrome b
• Defective chemotaxis, repiartory burst activity & bacterial
killing ….. Recurrent bacterial infection

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 Acquired functional disorder
Functional defect Disorder
Minimal •Neutrophil hyper segmentation due
to megaloblastic anemia
•Mucopolysaccaridoses
Adherence/ Movement/ •Renal failure
Migration •Diabetes •Neonates
•Malnutrition •Leukemia
Recognition •Immunoglobin deficiency
•Complement deficiency
Phagocytosis / killing •Malnutrition
•Vit. E deficiency
•Severe iron deficiency
•Neonates •Post-irradition
•Diabetes
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Neutrophil Alkaline phosphatase (NAP)
Principle:
• NAP is an enzyme present within the light granules
(phosphasomes) of maturing neutrophils
• Stimulated neutrophils contain increased amounts
of NAP, therefore, the test helps to distinguish
reactive neutrophilia (increased NAP) from
abnormally maturing clonal granulocytes of CML

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Procedure
• NAP usually determined semi quantitatively by
specific cytochemical staining of peripheral
blood smears
• The NAP present in the neutrophils hydrolyzes
the substrate that is then coupled to a dye
forming brown-to-black particles in the
cytoplasm of these cells at the enzyme sites
• These smears are then counter stained &
examined microscopically
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Score
200 neutrophils or bands are counted and graded 0 to
4+ by evaluating the number of cytoplasmic particles
& the NAP score is calculated by adding the products
of the number of cells multiplied by the grades
e.g. No. of cells X Grade Score
10 0 (no granules) 0
30 1 (very few granules) 30
30 2 (few to moderate) 60
20 3 (numerous) 60
10 4 (over crowded) 40

100 190
cells NAP score
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 Normal range: 35-100 (Dacie) with slight variation in
each lab
Specimen:
• Freshly prepared patient & control blood smears
• Smears should be dried at least 1 hour before fixation
• If not stained immediately, fixed slides may be stored
overnight in a freezer

N.B EDTA inhibits this reaction …. Unsatisfactory results

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NAP score (positive)

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A case of CML

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Interpretation:
Low score:
• CML (very low)
• Other hematopoietic neoplasms especially MDC
• PNH

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High score:
• Infections. Leukaemoid reaction
• MPD (not CML)
• Pregnancy
• Stress
• Oral contraceptive
• Drugs: lithium, corticosteroids, estrogen, CSF
• Aplastic anaemia
N.B the development of PNH in a patient with AA is associated
with low score
In CGl=CML: •Remission N or high score
•Infections, after splenectomy, 50% of cases of blastic crisis …. High score
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