WHITE BLOOD CELLS

profile and disorders

Linda D. Tamesis, MD
Dulce Ann Ross B. Dalangin, MD
 TYPES OF WBC
with non neoplastic disorders
 LYMPHOCYTE

⚫ Cellular immunity and

modulation of humoral immunity
⚫ CD4/CD8 = 2:1 (1-3.4)
⚫ Reference intervals:
Adult ≈1.5 to 4.0 × 109/L
Child ≈1.5 to 8.8 × 109/L
⚫ Survival
– Variable
LYMPHOCYTOSIS

⚫ Infectious Mononucleosis
⚫ Infections Lymphocytosis
⚫ Viral infections
⚫ Tuberculosis
⚫ Syphilis
⚫ Multiple Sclerosis
LYMPHOCYTIC DISEASES
Human T Lymphotrophic Virus

⚫ Infects and transforms T cells

⚫ Transmitted by breast milk or exposure to blood
⚫ Acute infection - fever, lymphadenopathy, skin rash
⚫ Progresses to adult T cell leukemia (HTLV-1) or tropical spastic
paraparesis
LYMPHOCYTIC DISEASES
Infectious Mononucleosis

⚫ Epstein-Barr Virus
⚫ Enters oropharynx and then B cells
⚫ Fever, pharyngitis, lymphadenopathy, atypical
lymphocytes (Downey cells)

⚫ DX- Paul-Bunnell heterophile antibodies (Monospot test),

VCA-Ab, EBNA-Ab
⚫ Risk of developing Hodgkin lymphoma, Burkitt
lymphoma, nasopharyngeal carcinoma
Atypical Reactive Lymphocytes
LYMPHOCYTOPENIA

⚫ Adults - below ≈1.8 × 10^9/L

⚫ Children - below ≈2.0 × 109/L

CAUSES
⚫ Radiation
⚫ Steroids
⚫ Carcinoma
⚫ HIV-AIDS
PLASMA CELL

⚫ Transformed B cell
⚫ Produces antibodies
⚫ Should not be seen in peripheral
circulation
 NEUTROPHIL

⚫ Phagocytosis of bacteria
⚫ Production
– 7-11 days
⚫ Survival
– 7-10 days
NEUTROPHIL

Distribution
⚫ Proliferating pool (bone marrow)
– myeloblasts, promyelocytes, myelocytes
⚫ Circulating pool (peripheral blood)
– Metamyelocytes (juvenile), bands,
neutrophils

Myelocyte - latest stage capable of cell division

NEUTROPHILIA
>8 x 109/L

Physiologic Pathologic
⚫ Hypoxia ⚫ Acute/Bacterial infections
⚫ Exercise ⚫ Acidosis
⚫ Stress ⚫ Snake bite
⚫ Epinephrine ⚫ MI
⚫ Smoking ⚫ Hemorrhage
⚫ Pregnancy ⚫ Diabetes
⚫ Burns
 NEUTROPHILIA

⚫ Child > Adult

– Adults - 1.8 to 7.0 × 10^3/μL
– Young children - 1.0 to 8.5 × 10^3/μL
⚫ Virulent > Benign
⚫ “shift to the left” - An increase in
immature peripheral blood
granulocytes
Band or Stab
Metamyelocyte
NEUTROPENIA < 2 x 109/L
Agranulocytosis/Severe neutropenia < 0.5 x 109/L

⚫ Severe sepsis
⚫ Megaloblastic anemia
⚫ Fanconi’s Anemia
⚫ Salmonella infection
⚫ Viral infection
⚫ Agranulocytosis
⚫ Radiation
 Hypersegmented
Neutrophil

Five-lobed nuclei in more

than 5% of the neutrophils
or any neutrophil with 6 or
more lobes
NEUTROPHILIC INCLUSIONS

TOXIC GRANULES
⚫ Severe inflammatory states,
infections
⚫ Azurophilic granules
⚫ Represent slower cytoplasmic
maturation
NEUTROPHILIC INCLUSIONS

DOHLE INCLUSION BODY

⚫ Light blue cytoplasmic inclusion
found at periphery
⚫ Remnants of free ribosomes or
rough endoplasmic reticulum
NEUTROPHILIC ANOMALIES

PELGER-HUET
⚫ Pince-nez
⚫ Granulocytes with bilobed nucleus
or band form
⚫ Autosomal dominant mutation in
lamin B-receptor gene
⚫ Count normal
⚫ Functionally normal
NEUTROPHILIC DISEASES
Chediak-Higashi Syndrome

⚫ Autosomal Recessive
⚫ Oculocutaneous albinism, frequent
pyogenic infections
⚫ Pancytopenia
⚫ Large granules in all WBC
⚫ Fusion of granules and vesicles
(such as lysosomes) leads to a
problem with phagocytosis
NEUTROPHILIC DISEASES
Chronic Granulomatous Disease

⚫ Autosomal recessive or X-linked primary

immunodeficiency
⚫ Defects in any of the components of NADPH
oxidase
⚫ Affects all phagocytic cells
⚫ Inability to kill intracellular microorganisms
⚫ Recurrent bacterial and fungal infections
⚫ Dx: nitroblue tetrazolium test, flow cytometry
 EOSINOPHIL

⚫ Phagocytosis of foreign particles

and immune complexes
⚫ Type 1 hypersensitivity reaction
⚫ Defense against helminthic
parasites
EOSINOPHILIA
> 0.5 X 109/L

⚫ Allergy
⚫ Parasitic infections
⚫ Skin disorders
⚫ Pernicious anemia
⚫ Loffler syndrome
(eosinophilic pneumonia)
 BASOPHIL

⚫ Type 1 hypersensitivity reaction

⚫ Delayed contact hypersensitivity
(cutaneous basophil hypersensitivity)
BASOPHILIA
> 0.2 x 109/L

⚫ Allergy
⚫ Chronic myeloid leukemia
⚫ Polycythemia vera
⚫ Hypothyroidism
 MONOCYTE

⚫ Phagocytosis of
mycobacterium, fungi,
protozoan, viruses, cellular
debris
⚫ Stimulates lymphocytic
response
MONOCYTOSIS
> 1.0 x 109/L

⚫ Recovery from acute infection

⚫ Mycotic, rickettsial, protozoal and viral
infections
⚫ Tuberculosis
⚫ Associated with hematologic neoplasms
 ? CYTOSIS

⚫ RELATIVE ⚫ ABSOLUTE
– % – WBC ct x %

Example:
WBC count = 15 x 10 9/L
Neutrophils = 60%
Is there a relative neutrophilia ?
Is there an absolute neutrophilia ?
LEUKOCYTOSIS
>11 X 109/L

PHYSIOLOGIC
⚫ Afternoon
⚫ After eating
⚫ Exercise

PATHOLOGIC
⚫ Infection
⚫ Inflammation
LEUKOPENIA
< 3 X 109/L

PHYSIOLOGIC PATHOLOGIC
⚫ Old age ⚫ Virus
⚫ Morning ⚫ Malignant disease
⚫ Pregnancy
INCREASED WBC ?

⚫ Physiologic - leukocytosis
⚫ Pathologic - leukemoid reaction

⚫ Neoplastic - leukemia
- lymphoma
 LEUKOCYTOSIS

LEUKEMOID LEUKEMIA
⚫ Reactive ⚫ Neoplastic
⚫ ≥ 50 x 10 9/L ⚫ ≥ 50 x 10 9/L
⚫ Immature and mature cells ⚫ Blastic and immature cells
⚫ Toxic granulations and Dohle ⚫ Spleen enlarged
bodies ⚫ LAP < 10
⚫ LAP > 10
LEUKEMOID REACTION
Leukemoid Reaction

⚫ A leukemoid reaction is an increase in the white blood cell

count, which can mimic leukemia.
⚫ Major causes: severe infections, intoxications, malignancies,
severe hemorrhage, or acute hemolysis
⚫ Blood counts often return to normal when the underlying
condition is treated.
Leukemoid Reaction

⚫ Increased WBC count >50x 109/L

⚫ Platelet count normal
⚫ Toxic granules presents
⚫ Dohle bodies present
⚫ Normal spleen
⚫ Increased LAP
⚫ Negative Philadelphia chromosome
LEUKEMIA
Hematopoietic Neoplasia

⚫ Disorders from hematopoietic stem cells of the BM or

lymphoid organs, assumed to be clonal processes due to
genetic errors
⚫ Differ in predominant cells, degree of differentiation, rates
of proliferation, clinical features, and response to therapy
⚫ Classified by morphology, clinical features, and location of
the abnormal proliferations
⚫ Now characterized at the level of the genetic abnormality
and the resultant intracellular pathway abnormalities.
Neoplastic Disorders of WBC

⚫ Chronic Myeloproliferative Diseases

⚫ Leukemia
⚫ Lymphoma
Leukemia: Signs and symptoms

⚫ Recurrent infections
⚫ Anemia
⚫ Easy bruising, excessive bleeding
⚫ Headaches
⚫ Bone pains
Leukemia – Risk factors

⚫ Myelodysplastic syndrome
⚫ Down syndrome
⚫ Bloom syndrome
⚫ Radiation
⚫ Tobacco smoke
⚫ Benzene
⚫ previous exposure to chemotherapeutic agents
 Acute Leukemia Chronic Leukemia
Age All ages Usually adults
Clinical onset Sudden Insidious
Course (untreated) 6 months or less 2-6 years
Leukemic cells >20% blasts More mature cells
Anemia Prominent Mild
Thrombocytopenia Prominent Mild
WBC count Variable Increased
Lymphadenopathy Mild Present; often prominent
Splenomegaly Mild Present; often prominent
MYELOGENOUS NEOPLASTIC DISEASES
Acute Myelogenous Leukemia

⚫ Affects all ages, but most common leukemia in older age

(>60 years)
⚫ Requires 20% blasts or more in blood or marrow for
diagnosis
⚫ Lymphadenopathy is rare
⚫ DIC is common
⚫ (+) Auer rods
Acute Myelogenous Leukemia
AML M3
APL Auer Rod
Auer Rod

• Eosinophilic rodlike structures in

cytoplasm of myeloblasts or
promyelocytes
• Derived from myeloperoxidase-
positive primary granules
Faggot Cell
AML Myelomonocytic. M4,M5
Acute Megakaryoblastic Leukemia (M7)
Chronic Myelogenous Leukemia

• young and middle-age, >50 years old

• Insidious, discovered incidentally
• anemia, weight loss, malaise, progressive splenomegaly – later
bleeding/bruising
• Laboratory features:
• WBC count >5x109/L and may exceed 30x109/L
– complete spectrum of granulocytic cells, <10% myeloblasts
– BCR-ABL present
Chronic Myelogenous Leukemia
blood
LYMPHOCYTIC NEOPLASIA
Acute Lymphocytic Leukemia

⚫ B or T lymphoblasts
⚫ <18 y/o
⚫ Symptoms: fever, night sweats
⚫ CNS involvement is common

ALL (blood)
ALL (bone marrow)
Chronic Lymphocytic Leukemia

M>F
>60 y/o
Insidious onset of fatigue, lymphadenopathy
Laboratory
• Thrombocytopenia, occasionally AIHA
• Small lymphocytes with condensed chromatin
• Cell surface markers: CD19, CD20, CD5, CD22, CD23, CD79a
Chronic Lymphocytic Leukemia
CLL bone marrow aspirate
Hairy Cell Leukemia

⚫ M>F, median age 50 years

⚫ Insidious onset with splenomegaly, leukopenia with monocytopenia
⚫ Medium-sized B cells with round to oval indented nuclei, reticular
chromatin, frayed cytoplasmic borders
⚫ Associated with other systemic immunologic disorders (Scleroderma,
Polymyositis, Polyarteritis nodosa)
⚫ BRAF mutation
HAIRY CELL LEUKEMIA

Abnormal B cells
with hair-like
projections
MULTIPLE MYELOMA

⚫ Neoplastic proliferation of plasma cells, occurring primarily in the BM

⚫ mean age, 62 y/o, M=F; rare <40 y/o
⚫ Sx: bone pain, punched out lesions in skull, pathologic fractures,
neurologic sx, metastatic calcification
⚫ Lab: marked degree of rouleaux formation; plasma cells, M-protein in
serum or urine

⚫ (hyperCalcemia, Renal insufficiency, Anemia, Bone lesions; CRAB)

MULTIPLE (plasma cell) MYELOMA (BMA)

Mott cell with Russel

body inclusions

Flame cell
MULTIPLE MYELOMA
MYCOSIS FUNGOIDES

• 2x frequently in men as in women, middle to late years

• Exfoliative dermatitis - plaques that form tumors that often ulcerate
• Biopsies of the skin - T cells with irregular nuclei show single-cell infiltration
of the epidermis and often form clusters known as Pautrier’s abscesses
⚫ Rare atypical mononuclear cells with cerebriform nuclei (Sézary cells) may
be present in the peripheral blood
MYCOSES FUNGOIDES
MYCOSIS FUNGOIDES
(T cell leukemia)

Sezary cells
with cerebriform
nucleus
WBC TESTS
Lab tests

⚫ CBC (differential)
⚫ PBS
⚫ LAP
⚫ Bone Marrow Aspiration and Biopsy
⚫ Fluorescent Flow Cytometry (CDs)
⚫ Cytogenetic analysis (FISH)
⚫ Chemistries: Uric acid, LDH, electrolytes
 Complete Blood Count

WHITE BLOOD CELLS RED BLOOD CELLS

⚫ Count ⚫ Count
⚫ Differential ⚫ Hemoglobin
⚫ Hematocrit
PLATELETS ⚫ MCV, MCH, MCHC
⚫ Count ⚫ RDW
⚫ Mean Platelet Volume
CBC - Automated

⚫ Cells are suspended in a fluid stream and their properties

are measured as they flow past sensors (flow cytometry)
⚫ Hydrodynamic focusing is used to isolate individual cells by
laminar flow.
Scatter plot
WHITE BLOOD CELL DIFFERENTIAL
In order to do a differential count, you must make
and stain a
SMEAR
 Materials

⚫ Blood, with EDTA, (from finger prick with red tip capillary
tube)
⚫ Microscope slide
⚫ 2 applicator sticks
⚫ Stain
Mix sample well.
Remove stopper.
Using a wooden applicator stick
rim the tube and check for
fibrin clots.
Place a 2-3 mm drop of
blood about 1/4 inch
from the end of the
slide (use two wooden
applicator sticks).
 Slide against the surface of the
first slide at an angle of 30 to 45
degrees and draw it back to
contact the drop of blood.
 Characteristics of a Good Smear

⚫ Thick at one end, thinning out to a smooth

rounded feather edge
⚫ Should occupy 2/3 of the total slide area
⚫ Should not touch any edge of the slide
⚫ No holes or gaps present
Poor smears
Good smear
 Increasing the angle of the
spreader slide will decrease
Adjustment of the length of the smear.
smear length Decreasing the angle will
increase the smear length.
Let smear air dry
completely before
staining
Once dry, staining
is done following
directions on
Coplin jars
⚫ Stain should be
an indigo color
and have a
metallic sheen

⚫ Color uniform
 Again let
smear dry
before viewing
under
microscope
 Place oil on the
feather edge and
view using oil
immersion lens
Ideal stain

⚫ No vacuoles in blood cells

⚫ RBCs are pink
⚫ Nuclei of WBC are purple
⚫ Eosinophilic granules are red-orange
⚫ Minimal precipitates
MANUAL
DIFFERENTIAL
Procedure

⚫ The slide is horizontally

moved to the next field
⚫ The procedure is repeated
until 100 leukocytes have
been counted (for a 100-
cell count).
Using a Schillings counter, classify
cells based on morphology
Included in the “normal” differential count

1) Neutrophil
2) Lymphocyte (reactive/atypical)
3) Monocyte
4) Eosinophil
5) Stab / Band
6) Basophil
7) Metamyelocyte /Juvenile
PERIPHERAL BLOOD SMEAR
PBS

⚫ Descriptive analysis of
– WBC
– RBC
– Platelets
PBS 10x

⚫ Assess quality of preparation

⚫ Assess RBC agglutination or rouleaux formation
⚫ Assess number, distribution and staining of leukocytes
PBS 40x

⚫ Find an area where RBC are evenly distributed/not distorted

⚫ Obtain a WBC estimate
WBC count estimation

⚫ Count the number of WBC in each of 10 high dry (40x) power fields.
⚫ Average the numbers.
⚫ Multiply the average by 2,000
(Average # WBC per 10 fields) x 2,000

Example: you see an average of 4 WBC in each high dry field

4 x 2,000 = 8,000 WBC/cc or 8 x 109/l

Descriptive analysis of WBC
100x

⚫ Predominant cell type

⚫ Level of maturity (shift to the left?)
⚫ Toxic change/reactive change
⚫ Abnormal forms/infectious agents
Normal

WBC
ESTIMATE
6,000 cm3
6 X 109/L
Increased
Decreased
BONE MARROW ASPIRATION/BIOPSY
FISH (Philadelphia Chromosome)
That’s All Folks!

Reference: Henry’s Clinical

Diagnosis and Management by
Laboratory Methods 24th Edition