2. LEUKOCYTES STRUCTE AND
FUNCTIONS
• Function
– Protect the host from infectious agents or
pathogens
• Innate=Natural Immune response
– 1st response to pathogens (non-self)
– Always available
– Neutrophils, monocytes, and macrophage main players
• Adaptive Immune response
– Starts in lymphoid tissue
– Slow
– Long-lasting immunity (memory)
– Lymphocytes become “activated
3. Myelopoiesis and Lympopoiesis
– Myelopoiesis: production and development
of myeloid cells in the bone marrow; Also
known as granulocytopoiesis.
– Lympopoiesis: production and developmet of
lympoid cells in the bone marrow.
4.
5.
6. Maturation of the granulocytic series
**immature to mature**
• Myeloblast
– Size: 14-20µm
– Cytoplasm
» Slight
amount/blue
» No granules
(possible Auer
rods to be
discussed in
another section)
– Nucleus
» Round/Central
or eccentric
» Reddish purple
» 1-3 nucleoli
» Fine
meshlike/lacy
chromatin with
no clumping
– N:C ratio = 4:1
7. Maturation of the granulocytic series
• Promyelocyte (progranulocyte)
– Size: 15-21 µm
– Cytoplasm
• Increased amount/blue
• Fine, azurophilic,
nonspecific granules present
– Nucleus
• Round/Central or eccentric
• Reddish-purple
• Fine, meshlike/lacy
chromatin beginning to
clump
• 1-2 nucleoli
– N:C ratio = 3:1
8. Maturation of the granulocytic series
• Myelocyte
– Last myeloid cell capable of
division
– Size: 12-18µm
– Cytoplasm
• Increased, bluish-pink
(“dawning of neutrophilia”)
• Fine specific granules
• Secretory vesicles
– Nucleus
• Round or oval/eccentric
• Reddish-purple
• Chromatin loosely clumped
• No nucleolus
– N:C ratio = 2:1 or 1:1
9. Maturation of the granulocytic series
• Metamyelocyte
– Also called “meta” or “juvenile”
– Size: 10-18µm
– Cytoplasm
• Moderate amount
• Specific granules
(neutrophilic, eosinophilic or
basophilic)
• Tertiary granules
• Secretory vesicles
– Nucleus
• Indented (kidney-bean
shaped)
• Central or eccentric
• Purple
• Clumped chromatin
• No nucleolus
– N:C ratio = 1:1
10. Maturation of the granulocytic series
• Band
– 1st stage found in peripheral
blood
– Size: 9-15µm
– Cytoplasm
• Moderate amount
• Specific granules (see meta)
– Nucleus
• Elongated, narrow band
(sausage) shape with
uniform thickness
• Central or eccentric
• Clumped, coarsely granular
chromatin
11. Maturation of the granulocytic series
• Segmented neutrophil
– Size: 10-16µm
– Cytoplasm
• Neutrophil - Granules stain
equally with eosin and
methylene blue, causing a
pinkish appearance
– Normal in circulation
= 40 - 80%
– Nucleus
• Neutrophil - purplish-red,
clumped granular
chromatin, 2-5 distinct
nuclear lobes connected by a
filament of chromatin
12. Eosinophils
Eosinophil - Granules
stain mainly red, due to
reaction with eosin in
stain
Normal in circulation = 0-
5%
Eosinophil - deep
blue purple,
coarsely granular
chromatin, 2
distinct lobes
14. Basophils
Basophil - Granules stain
mainly blue, due to the
reaction with methylene blue
in stain
Normal in circulation = 0-
1%
Mast cell is a tissue
basophil
Basophil - deep blue purple,
coarsely granular chromatin, 2
nuclear lobes (sometimes
obscured by cytoplasmic
granules
16. Function of the granulocytic cells
• Neutrophils
– Defense against bacterial infections
• Eosinophils
– Regulate inflammation
– Respond to antigenic stimulation in chronic allergies
– Interact with larval stages of some helminthic parasites
• Basophils
– Histamine in granules plays a role in acute, systemic allergic
reactions (sudden release of histamine can cause anaphylactic
shock)
– Granules also contain heparin
17. Monocytes
• Monopoiesis
- the production and development of
monocytes, macrophages and their
precursors. Monocytes are produced in
the bone marrow.
18. Monocytes cont’
• Differentiation & maturation
– Produced from CFU-GM
– Can further differentiate into macrophages in the tissues
• Lifespan
– P.B ~ 8 hours
– Tissue
• Regulation
– Production primarily regulated by cytokines
• GM-CSF
• IL-3
• M-CSF
20. Maturation of monocytic series
• Monoblast
– Size: 12-20µm
– Cytoplasm
• Blue, moderate amount
• No granules
– Nucleus
• Round, oval or slightly folded
• Eccentric
• Reddish-purple
• Chromatin fine and lacy
• Vacuoles may be present
• 1-2 nucleoli
– N:C ratio = 4:1
21. Maturation of monocytic series
• Promonocyte
– Size: 12-20µm
– Cytoplasm
• Pale gray-blue and abundant
• “Bleb-like” pseudopods at periphery
• May contain fine red “dust-like” particles, azurophilic granules
• Vacuoles may be present
– Nucleus
• Round with chromatin creases or brain-like folding
• Central
• Reddish-purple with light clumping of chromatin
• 0-2 nucleoli
– N:C ratio = 3:1 or 2:1
22. Maturation of monocytic series
• Mature monocyte
– Normal in circulation: 2-9%
– Size: 12-20µm
– Cytoplasm
• Pale gray-blue and abundant
• Bleb-like pseudopods at periphery, variable shape
• Numerous fine pale red dustlike granule scattered throughout
• Vacuoles common
– Nucleus
• Increased folding or elongated
• Central
• Purplish with finer clumped chromatin
• No nucleoli
– N:C ratio = Variable
23. Machrophages
• Once the monocyte enters the tissues, it becomes a macrophage
– Size:15-80µm
– Cytoplasm
• Gray-blue and abundant
• Ragged edges
• Vacuoles common
– Nucleus
• Round with netlike appearance
• Purplish with finer clumped chromatin
• Nucleoli appear
24. Macrophages in various tissues
• Macrophage alias’
– Histiocytes in the loose connective tissue
– Kupffer cells in the liver
– Osteoclasts in the bone
– Microglial cells in the nervous system
– Also scattered throughout the body in all
organ tissues (spleen, lung, abdomen,
26. Lymphocytes
• Lymphopoiesis
The production and development of lymphocytes and their
precursors.
• Primary lymphoid tissue
• Bone marrow
• Secondary lymphoid tissue
– Lymph node, spleen, Peyer’s patches in intestine and
mucosal tissues such as tonsils
– Active immune response occurs in above tissues where
lymphs communicate and interact with antigen-presenting
cells, phagocytes and macrophages
27. Lymphocyte differentiation and
maturation
• Develop from HSC (hematopoietic stem cell)
• Common lymphoid progenitor cell (CLP) gives rise to
the committed precursor cells:
T lymphocytes (60-80% of lymph population)
• thymus
B lymphocytes (10-20%)
• Bone marrow
Natural killer cells (NK)
30. Lymphocyte population and function
– T Lymphocyte Function
• Cellular immune response (they do not produce antibodies)
• Three subsets
– Helper T cells
» Instrumental in aiding B cells in antibody production
– Suppressor T cells
» Act as a “thermostat” to shut off the system or keep it under
control
– Cytotoxic T cells
» Destroy by lysis of target cells (such as tumor cells)
» Play a role in inflammatory response
» Host defense when stimulated by interferon against certain
viruses (cytomegalovirus - CM, and hepatitis) by killing the
virally infected target cell before the virus replicates
• Responsible for chronic organ graft rejection. (T cells have many unique
antigens on their cell surfaces, some of which are the HLA - human
lymphocyte antigen - markers. HLA markers are instrumental in chronic
graft rejection and that is why tissue typing is done to establish a good
donor match.)
31. Lymphocyte population and function
cont’
• B Lymphocyte Function
• Humoral immune response by transforming into
antibody-producing plasma cells
• Defense against encapsulated bacteria such as
Streptococci
• Mediate hyperacute organ rejection
32. Lymphocyte population and function
cont’
• Natural killer (NK) cells Function
– Recognize and kill tumor cells
– Recognize and kill cells infected with a virus
34. Maturation of lymphocytic series
• Lymphoblast
– Cell size: 10-18µm
– Cytoplasm
• Blue/scanty
• No granules, Auer rods
are never present
– Nucleus
• Purple, smooth
chromatin
• Smudged
• Round, central or
eccentric
• 1-2 nucleoli
– N:C ratio = 4:1
35. Maturation of lymphocytic series
• Prolymphocyte
– Size: 9-18µm
– Cytoplasm
• Blue, scanty
• Usually granules are absent,
but a few azurophilic granules
may be present
– Nucleus
• Purple, condensed chromatin
• Round or indented, eccentric
• 0-1 nucleoli
– N:C ratio = 3:1
36. Maturation of lymphocytic series
• Mature lymphocyte
– Normal adult value: 25-35%
• Cell size: 7-10µm (a typical normal
lymph has a nucleus that is the
size of a normal RBC)
– Cytoplasm
• Light blue, scanty to moderate
• Few azurophilic granules may be
present
– Nucleus
• Purple, dense, clumped chromatin
• Round or indented, eccentric
• No nucleoli
37. Large lymphocytes versus monocytes
• Size
– Large lymph: 12-15µm
– Mono: 15-18µm
• Nucleus
– Large lymph: clumped, condensed
– Mono: lacy, brainlike folds
• Granules
– Large lymph: large azurophilic, easy to count
– Mono: red, fine
• Cytoplasm
– Large lymph: clear to light blue color, may be indented by red cells
– Mono: “ground glass” appearance, projection of blunt pseudopod blebs
40. Plasma cells
• Plasma cells
– Function is the synthesis and excretion of antibodies
(immunoglobulins)
– Normally not present in the peripheral blood; comprise 2% of bone
marrow cells. (May be seen in the peripheral blood in the disease
called multiple myeloma, a disease of uncontrolled production of
immunoglobulins.)
– End stage of the B lymphocyte
– Appearance
• Size: 10-18µm
• Cytoplasm is dark blue with perinuclear halo and may contain
vacuoles indicating antibody synthesis
• Nucleus is round, eccentric, dark purple with dense clumped
chromatin
– Variant plasma cells
• Grape or Mott cell - cytoplasm completely filled with red, pink or
colorless globules called Russell bodies
• Flame cell - cytoplasm stains bright red-staining proteinaceous
material
41. Non-malignant disorders of Leukocyte
• Affect granulocytes and monocytes
• Response to various non-malignant
disease states and toxic changes
• Changes can be qualitative or quantitative
42. Important terms
• Leukocytosis
– Total leukocyte count is more than 11.0 x
109/L in an adult
– Most commonly caused by increase in
neutrophils
• Leukopenia
– Decrease in leukocytes below 4.5 X 109/L
– Most commonly caused by decrease in
neutrophils
43. Review: Neutrophil Function
• The contents of primary, secondary and
tertiary granules in neutrophils are enzymes
which are involved in killing and digesting
bacteria and fungi.
44. Review: Neutrophil Function
Cont’
• Primary function is phagocytosis which
occurs in three stages.
– Stage 1: Migration and diapedesis
• Chemotaxis is the process of directional migration
which occurs under the guidance of
chemoattractants which are produced by the site of
injury.
• The neutrophil transforms from smooth and round
to rough and flat.
• Diapedesis is the movement of the neutrophil
through the vessel wall.
45. Review: Neutrophil Function
Cont
–Stage 2: Opsonization and recognition
• Opsonization is the mechanism which
facilitates recognition and attachment to the
organism to be ingested.
• After the bacterium is coated by
immunoglobulin and complement, it is
referred to as an opsonin.
46. Review: Neutrophil Function
Cont
– Stage 3: Phagocytosis: Ingestion, killing and
digestion
• The cytoplasm of the neutrophil forms a
pseudopod which surrounds and envelops the
microorganism forming a vacuole called a
phagosome.
• Cytoplasmic granules migrate to the vacuole
and release their lytic contents which kill and
digest the organism.
47. Disorders of Neutrophils:
Quantitative
• Causes
– Malignant
• Neoplastic transformation of hematopoietic
stem cells (discussed later)
– Benign
• Acquired
• Neutrophilia: increase in total circulating
absolute neutrophil concentration
• Neutropenia: dicrease
48. Neutrophilia
• ANC > 7.0 x 109/L
• Occurs as a result of a reaction to a pathologic or physiologic process
(reactive neutrophilia)
– Immediate
• Increase lasts about 20-30 minutes
• Redistribution of neutrophils from marginal pool to circulating pool
• Neutrophils are mature
• Seen in acute exercise, anxiety
• “shift neutrophilia” or “pseudoneutrophilia”
– Acute
• Occurs 4-5 hours post-pathologic stimulus (i.e bacterial infection)
• Increase in flow of neutrophils from the bone marrow pool to the blood
• Immature neutrophils numbers may increase
– Chronic
• Follows acute neutrophila, if the stimulus continues beyond a few days
• Storage pool in bone marrow depletes
• Bone Marrow shows increased numbers of early neutrophil precursors
• “shift to the left”
49. Conditions associated with
neutrophilia
• Reactive chronic neutrophilia
–Leukocytes < 50 x 10 9/L
–Shift to the left
–Presence of toxic granulation,
DÖhle bodies and cytoplasmic
vacuolization
51. Neutrophilic conditions
• Bacterial Infection
– Most common cause of neutrophilia
– Seen with staphylocci and streptococci infections
– Bone marrow increases output of storage neutrophils to peripheral
blood, see shift to the left
• Physiologic leukocytosis
– No shift to the left
– Birth to the first days of life
– Childbirth
– Extreme temperatures
– Emotional stimuli
• Tissue destruction/Injury, Metaboloic disorders
– Neutrophil input is increased from the bone marrow to the tissue
– Examples include: Rheumatoid arthritis, burns, gout, uremia, trauma
52. Neutrophilic conditions
• Leukoerythroblastic reaction
– Presence of RBCs
– Shift to the left
– Poikilocytosis, tear drops, anisocytosis
– Associated with chronic neoplastic myeloproliferative
conditions
53. Neutrophilic conditions
• Leukemoid reaction
– Leukocytes> 50 x 10 9/L
– Advanced degree of leukocytes in the
blood that is not a result of leukemia
– Transient; leaves when stimulus is
removed
– Many circulating immature leukocyte
precursors seen
– Seen in chronic infections, carcinoma of
certain organ systems
– Blood picture similar to chronic
myelocytic leukemia(CML)
• Leukocyte Alkaline Phosphatase, is
used to differentiate leukemoid
reaction from CML
• LAP increased in leukemoid reaction,
decreased in CML
54. Neutropenia
• ANC <1.5 x 109/L
– Causes
• Increased cell loss
– Increased neutrophil diapedesis (Tissue egress)
– Bone marrow can not keep up with cell utilization
– Examples: immune neutropenia, hypersplenism
• Megaloblastic Anemia
Hypeplastic bone marrow
Abnormal myeloid cells destroyed
• Decreased bone marrow production
– M:E ratio decreased
– Myeloid hypoplasia
– Storage pool in bone marrow decreased, as is circulating and
marginal pool
– Examples: stem cell disorders, aplastic anemia,
chemicals/drugs
55. Cytoplasmic inclusions
Inclusion Characteristic Composition Conditions
Döhle body Light gray-blue
oval near periphery
Rough
endoplasmic
reticulum (RNA)
Infections, burns,
cancer,
inflammatory states
Toxic granules Large blue-black
granules
Primary granules Same as above
Cytoplasmic
vacuole
Clear, unstained
circular area
Open spaces from
phagocytosis
Same as above
Bacteria Small basophilic
rods or cocci
Phagocytized
organisms
Bacteremia or
sepsis
Fungi Round or oval
basophilic
inclusions, larger
than bacteria
Phagocytized
organisms
Fungal infections
Morulae Basophilic,
granular, irregular
Clusters of Ehrlichia
rickettsial organims
Ehrlichiosis
56. Nuclear abnormalities
Anomaly Description Conditions
Pelger-Huët •Neutrophil nucleus is
bi-lobed or has no
lobes
• May have a “pince-
nez” appearance
• clumped chromatin
•Can be real or pseudo
caused by drug
ingestion or leukemia
Hypersegmentation •larger than normal
neutrophils with 6 or
more nuclear lobes.
•Megaloblastic anemia
Pyknotic nucleus •Degenerating nucleus •Dying neutrophils
57. Inherited functional
abnormalities
Condition Morphologic or
Functional Defect
Clinical Features
Alder-Reilly •Large, dark cytoplasmic
granules in all
leukocytes
•Cells function normally
Associated with
mucopolysaccharidosis
Chediak-Higashi
Syndrome
•Giant fused granules in
neutrophils and lymphs
•Cells engulf but do not
kill microorganisms
Often fatal due to
recurrent pyogenic
infections
May-Hegglin •Blue, Döhle-like
cytoplasmic inclusions in
granulocytes
•Cells function normally
•Bleeding tendency from
thrombocytopenia
•Giant platelets
Chronic granulomatous
disease
•Defective respiratory
burst
•Cells engulf but do not
•Recurrent infections,
esp. in childhood
•Prognosis poor
59. Quantitative disorders
• Monocytosis
–AMC > 0.8 x 109/L
–Seen in inflammatory conditions
and malignancies
• Monocytopenia
–AMC < 0.2 x 109/L
–Seen in stem cell disorders
60. Qualitative disorders
• LIPID (LYSOSOMAL)STORAGE DISEASES
– Inherited disorders
– Accumulation of un-metabolized material in
the lysosomes of various cells. They are
caused by various enzyme defects (in-born
errors) in lipid metabolism linked to an
enzyme deficiency
– Three main disorders
61. Disorders I
• Gaucher’s Disease
– Enzyme deficiency: β-glucocerebrosidase
– Prevalent in the Ashkenazi Jewish population
(eastern European)
– Macrophage can not digest the stroma of
ingested cells
– Results in an accumulation of
glucocerebroside
62. Gaucher’s cells
• Large histiocyte (20-
100 µm)
• Displaced nucleus
which contains one or
more round nucleoli
• Cytoplasm is faintly
blue/white with
characteristic
“crumpled tissue paper”
appearance which is
probably the result of
glycolipid deposition
63. Disorders II
• Niemann-Pick Disease
– Enzyme deficiency: sphingomyelinase
– Increased incidence in the Jewish population
– Causes an accumulation of un-metabolized
sphingomyelin and cholesterol
– Classic form presents with jaundice at birth,
hepatosplenomegaly, enlarged lymph nodes,
neurological symptoms, retarded physical and mental
development. Death occurs by age 3.
65. Disorders III
• Tay Sachs Disease
– Enzyme deficiency: hexosaminidase A
– Higher incidence in Ashkenazi Jewish population
– Severity of the disease correlates with residual enzyme
activity.
– The buildup of un-metabolized GM2 (Ganglioside
monosialic acid)
– ganglioside in the tissues has devastating effects in the
central nervous system and eyes.
– Physical and mental deterioration occur along with
seizures and paralysis. Death comes by age 4.
66. Lymphocytes disorders
• Lymphocytes originate primarily from bone
marrow and thymus
• Secondary organs include spleen, lymph
nodes, tonsils, and Peyer’s patches in GI tract
• 3 general populations
– B- lymphs: 10-20 %
– T-lymphs: 60-80%
– NK: < 10%
67. Antigen-stimulated lymphocytes
• Reactive or Atypical: Atypical is widely used; however, connotes abnormal or
malignant
• Downey Cell: obsolete term for reactive lymph and immunoblasts
• Immunoblasts:
– large cells with prominent nucleoli
– fine clear chromatin
– dark purple-blue cytoplasm
– preparing for or engaged in mitosis in response to stimulus
• Plasmacytoid lymphs:
– daughters of B immunoblasts
– Eccentric nucleus with moderate amount of deep blue cytoplasm
• Plasma Cell
– Fully differentiated B cell
– Eccentric nucleus with “cartwheel appearance” with large amount of
basophilic cytoplasm
– Perinuclear clearing (Golgi apparatus)
– Releases Ig
68. Reactive
Lymphocyte
Normal
Lymphocyte
Size 9-30 µm 8-12 µm
N:C ratio Decreased High
Cytoplasm Abundant
Colorless to dark
blue
Azurophilic
granules
Can scallop the
RBCs
Scant
Colorless to light
blue
Nucleus Elongated,
irregular
Round
Chromatin Coarse to
moderately fine
Coarse
69. Benign lymphocyte disorders
• Majority of disorders affecting
lymphocytes are acquired
– Hallmark: reactive lymphocytosis
– Reactive process
• Congenital disorders
– Defect is found within lymphocytic system
70. Benign lymphocyte disorders
Cont’
• It is important to differentiate benign conditions
associated with lymphocytosis from malignant
lymphoproliferative disorders
– How?
• Presence of heterogeneous reactive lymphs
• Positive serological test for antibodies against
infectious organisms
• Absence of anemia and thrombocytopenia
• All of above favor a benign diagnosis
71. Lymphocytosis
• Excess of lymphocytes in the blood.
• Absolute lymphocyte count (ALC) > 4.8 x 109 /L
in adults
• Relative count > 35-45%
• Self-limited
• Reactive process is due to infection or
inflammatory conditions
• B and T cells involved
• Lymphocytes develop in response to antigenic
stimulation. They become “activated”
72. Causes of reactive
lymphocytosis
• Infectious mononucleosis (IM)
– Caused by the Epstein-Barr Virus (EBV) which enters the body via
saliva (“kissing disease”)
– EBV Pathophysiology
• EBV attaches to B lymphs by receptor CD21 which causes it to
express the activation marker CD23 that stimulates B-lymphocyte
growth factor.
• The virus is incorporated into the lymph genome making the cell
express viral proteins on the cell membrane and immortalizes the
line of EBV-lymphs.
• Activated cytotoxic T-lymphs are released to inhibit the activation
and proliferation of EBV infected lymphs. These are the
characteristic Reactive Lymphs.
– Clinical symptoms
• Classic triad: fever, pharyngitis and lymphadenpathy
• Dysphagia (difficulty swallowing)
• General malaise
• Fatigue
• Spleen is enlarged and nodes are firm but not tender or warm
– Generally seen in children and young adults (14-24 yrs old
73. Laboratory features of IM
– CBC
• Relative lymphocytosis
– Peaks at 2-3 weeks of infection, remains elevated for 2-8
weeks
• Leukocyte count 12-25 x 109/L
• Peripheral smear
• Reactive lymphocytes , historically referred to as a Downey cell
with irregular cytoplasmic border, increased cytoplasm and
dark blue edge around the periphery of the cytoplasm.
• >20% reactive lymphs
– Serologic test
• Heterophil antibody test (i.e Monospot)
74. Causes of reactive
lymphocytosis
• Toxoplasmosis
– Infection with intracellular protozoan
Toxoplasma gondii
– Acquired infections in children and adults
due to ingestion of oocysts from cat feces or
undercooked meat
– Can be transmitted via placenta
75. Causes of reactive
lymphocytosis
• Cytomegalovirus (CMV) Infection
– Belongs to herpes family
– Endemic worldwide
– Acquired through transfusions, sexual contact
and close contact
– Can be transmitted across placenta
– Poor prognosis for immunocompromised
individuals who contract virus
76. Causes of reactive
lymphocytosis
• Infectious lymphocytosis
– Affects children
– Viruses include Adenovirus, Coxsackie A and
Bordetella pertussis
– Leukocytosis and lymphocytosis occur in first
week of illness then return to normal
77. Lymphocytosis: laboratory
testing
• Tests include CBC, serology, and microbiologic
cultures. The most useful tests are still the CBC
with differential and serology tests.
• Proper evaluation of peripheral blood smear
crucial for correct diagnosis of absolute
lymphocytosis - especially for patients with
infectious mononucleosis.
• Serology tests important, especially the Monospot
test for infectious mononucleosis. A positive
Monospot and a differential with reactive
lymphocytes is diagnostic for infectious
mononucleosis in some labs.
• Further serology testing differentiates among
variety of viral infections.
78. Lymphocytopenia
• Absolute lymphocyte count < 1.0 x 109/L
• Causes
– Decreased production or increased destruction of lymphocytes
– Changes in lymphocyte circulation patterns
– Corticosteroid therapy
79. Immune deficiency disorders
• Impaired function of one or more of the
components of the immune system: T, B,
or NK lymphocytes
• Body unable to mount an adaptive
immune response
• Can be acquired or congenital
80. Acquired immunodeficiency
• Acquired immune deficiency syndrome
(AIDS)
– Infection with a retrovirus, human
immunodeficiency virus type-1 (HIV-1)
– Transmission through sexual contact or
contact with blood and/or blood products
– Binds CD4 antigen on helper T lymphocytes
which results in cell lysis
81. Congenital immunodeficiency
• Decrease in lymphocytes and impairment
in either cell-mediated immunity (Tcells),
humoral immunity(Bcells) or both
• Lymphocytes appear normal on the
peripheral smear
82. Congenital immunodeficiency
• Severe Combined immunodeficiency Syndrome
– Major qualitative immune defects involving both
humoral and cellular immune functions
– Fatal by 2 years if untreated by bone marrow
transplant or gene therapy
• Wiskott-Aldrich Syndrome
– Patients have recurrent infections due to
immunodeficiency (decreased CD8 T-cells),
thrombocytopenia and eczema
83. Introduction to Haematological
malignancies Or neoplasm
• Important terms:
• Neoplasm or tumor: “New growth”
– Results from a dysregular prolieferation of a single transformed cell
– Can be malignant or benign
• Malignant
– “Deadly”
– Clone of abnormal , proliferating cells, without function or
differentiation
– Have the potential to metastasize or get bigger
• Benign
– Premalignant
– Originate from highly organized, differentiated cells
– Do not spread or invade surrounding tissues
84. • Cancer: A malignant tumor; growth not encapsulated;
infiltrates into surrounding tissues; spreads by lymphatic
vessels to other parts of body; death caused by destruction of
other organs, by extreme debility and anemia or by
hemorrhage.
• Carcinogenesis: Complex multistage process by which cancer
develops.
• Oncogenic: causing a tumor
• Metastases: Secondary tumors derived from cells of primary
tumor that disseminated to other parts of the body.
• Leukemia: A malignant disease of hematopoietic tissue
characterized by replacement of normal bone marrow
elements with abnormal (neoplastic) blood cells. Abnormal
cells are also seen in the peripheral blood
• Lymphoma: Abnormal proliferation of lymphoid cells within
the lymphatic tissue or lymph nodes, results in a solid tumor
85. Characteristics of cancer
• Alterations in the expression of multiple genes
• Dysregulation of normal cell division and
differentiation
• Imbalance between proliferation and
apoptosis
86. Events needed to cause malignant
transformation
• Increased cell growth
• Impairment of differentiation
• Altered relations with the microenvironment
• Impairment of programmed cell death
87. What makes Cancer “Cancer”?
1. decreased requirements for growth factors
and serum
2. are no longer anchorage dependent
3. grow independently of density
Normal cells:
eventually enter Go
confluent monolayer CHECKPOINT FAILURE
contact inhibition
90. Classification of Neoplasms in the
Bone Marrow
• Lymphoid
– Only lymphocytic cells affected
• Myeloid
– Granulocytes, monocytes, megakaryocytes,
erythrocytes affected
• Both Lymphoid and Myeloid lines can include
benign and malignant neoplasms
93. General Classification
Acute Chronic
Age All ages Adults
Clinical onset Sudden Gradual
Course of disease Weeks to months Months to years
Predominant cell Blasts
Some mature forms
Mature forms
Anemia Mild-severe Mild
Thrombocytopenia Mild-severe Mild
WBC Variable Increased
Organomegaly Mild Prominent
95. How Do Leukemias Arise?
• Somatic mutation of a
single hematopoietic
stem or progenitor cell
• Unlimited self-renewal
of the cancer-initiating
cell
• As the mutant cell line
predominates, normal
hematopoiesis is
inhibited causing
leukemic cells to spill
into peripheral blood
96. Proto-oncogenes and Oncogenes
• Proto-oncogene: normal unaltered gene that has the
potential to become an oncogene. It is a cellular
counterpart which can be turned into an oncogene.
• Oncogene: altered cell genes that cause tumors. This is
a “cancer gene”; often found in viral genomes.
• Located at breakpoints of chromosomal aberrations,
such as translocations
103. Epidemiology
• Most new cases found in older adults ( > 67 yrs old)
• 50% of leukemias are acute
• More common in whites
• Age groups
– ALL: children 2-5 years old; lymphoid
– CLL: Adults > 50; lymphoid
– AML: adults; myeloid
– CML: adults; myeloid
104. Clinical Findings
• Anaemia
– Due to erythropenia
• Infections
– Due to neutropenia
• Bleeding Episodes
– Due to thrombocytopenia
• Bone pain
– Due to marrow expansion
• Weight loss
105. Evaluation of Leukemia
• Note the onset of symptoms
• Analyzing CBC
– Thrombocytopenia?
– Red cell count/anemia?
• Observe cell lineage
– Lymphoid or Myeloid?
• Assess maturity of predominating cells
106. Laboratory Features
• Normochromic, normocytic anemia
• Thrombocytopenia
• Platelet morphology and function can be
abnormal
• Leukocyte count can be increased, decreased or
normal
• Immature leukocyte precursors are seen
• Bone marrow is hypercellular
• Maturation abnormalities in all cell lines
• Uric acid is increased
107. Official Classification of Hematopoietic
Neoplasms
• Two systems
– French-American British (FAB)
• Historical
– World Health Organization (WHO
• Widely accepted
• Important because..
– Allows clinicians a way to compare therapeutic regimens
– System for ID and comparison of clinical features & lab findings
– Permit associations of cytogenetic abnormalities with disease
108. FAB classification
• Consists of three groups
– Myeloproliferative Disorders(MPD)
– Myelodysplastic Syndromes(MDS)
– Acute leukemia (AL)
• Based on morphological characteristic of
Wright-stained cells in peripheral blood or
bone marrow with supplementary
cytochemical stains
109. WHO Classification
• Classification system uses morphology, cytochemistry and
immunophenotyping to determine cell lineage and degree of maturation
(similar to FAB)
• Additionally uses genetic and clinical features prior to therapy and history
of MDS to define subgroups
• Consists of four groups
– Myeloid
• Further classified as MPD, MDS/MPD, MDS, AML
– Lymphoid
• Further classified as B cell, T/NK cell, Hodgkin’s Disease
– Mast cell
– Histiocytic
110. Laboratory Techniques for Diagnosis
and Classification of Neoplasms
Cytochemical analysis
– In vitro staining of cells to look at their chemical
composition
– Evaluation of positivity in these stains must be determined
on the leukemic blast stage of the cell
– Usually performed on bone marrow slides
– Helpful in differentiating lymphoid or myeloid lineage of
blasts in AL
– Reactions are either enzymatic or non-enzymatic
111. Cytochemical stains
• Types
– Myeloperoxidase (MPO)
• Activity is present in the primary granules and Auer rods of
myeloid cells
• Separates myeloid and lymphoid blasts
• Stains late myeloblasts, granulocytes, monocytes less intensely
• Differentiates AML from ALL
• Granules stain reddish-brown
• Smears must be fresh
112. Cytochemical stains – con’t
– Sudan Black B
• Activity is present in phospholipids in the membrane of 1̊ (non-specific)
and 2̊ granules (specific)
• Parallels myeloperoxidase but smears do not have to be fresh
• Granules stain black
– Periodic Acid Schiff (PAS)
• Activity is in glycogen and related substances
• Stains lymphocytes, granulocytes, megakaryocytes
• Helpful in diagnosing erythroleukemia where there is strong reactivity in
normoblasts
• Stains red-purple in blocks in cytoplasm
113. Cytochemical stains – con’t
• Esterases
– Specific Esterase (Naphthol AS-D Chloroacetate)
• Activity is in cytoplasm
• Stains neutrophilic granulocytes, differentiates monoblasts and
myeloblasts
• Granules of myeloblasts stain blue-black
– Nonspecific Esterase (Alpha-Naphthyl Acetate)
• Activity is in cytoplasm
• Stains monocytes and also megakaryocytes
• Differentiates myeloblasts from monoblasts (can use a double staining
technique to view both specific and non-specific stains on one smear)
• Addition of Na fluoride to this stain inhibits activity in monocytes
• Granules stain orange-red
114. Cytochemical stains – con’t
• Leukocyte Alkaline Phosphatase (LAP)
– Enzyme within the 2O or specific granules of
maturing granulocytes
– Distinguishes leukemoid reactions ( ) from chronic
myelogenous leukemia ( )
• Acid Phosphatase
– Present in lysosomes in normal leukocytes
– Helpful in diagnosing hairy cell leukemia
115. Laboratory Techniques for Diagnosis
and Classification of Neoplasms
Immunologic marker studies
– Cell surface markers
• Monoclonal or polyclonal antisera added to cell
suspensions of fresh peripheral blood or bone marrow
and an immunofluorescent method is used in a flow
cytometry instrument to analyze the markers which are
expressed as cluster designations (CD).
• CDs identify antibodies that are specific for certain cells
and allow for a positive identification.
116. Laboratory Techniques for Diagnosis
and Classification of Neoplasms
Molecular Genetics
– This newer method of diagnosing leukemia consists of DNA probes and
polymerase chain reaction (PCR)-based studies. They are rapid and precise
and are used to confirm chromosomal abnormalities that are not detected by
conventional studies. They are also used to monitor residual disease following
therapy.
– Cytogenetics (Chromosome studies)
• Identifies chromosome translocations which are specific for
certain leukemias
• Philadelphia chromosome (t[9:22]) is associated with CML
• t[15:17] is associated with acute promyelocytic leukemia
117. Treatment
• Cures are not common except in childhood
leukemia. The best hope for a cure in adults
lies in bone marrow transplantation.
– Cytoreductive chemotherapy
• Reduces the leukemic cell mass
• Blocks DNA synthesis
• Blocks RNA synthesis
• Complications arise from marrow hypoplasia and the
resulting cytopenia
118. Treatment
– Radiotherapy (radiation)
• Kills focalized leukemic cells
• Usually used in addition to chemotherapy and for CNS prophylaxis
– Bone marrow transplantation
• Bone marrow is eradicated with chemo and radiation.
• Compatible donor cells are transfused and they travel to the
empty marrow where they engraft and repopulate the marrow
with healthy cells.
• Complications include graft vs host disease (GVHD) which can be
fatal.
121. 1. Overview of AML
• Also known as
- Acute myelocytic leukemia
- Acute myelogenous leukemia
- Acute non-lymphocytic leukemia
• Stem cell disorder characterized by malignant
neoplastic proliferation and accumulation of
immature and non-functional hematopoietic cells in
the bone marrow
• Neoplastic cells show increased proliferation
and/or decreased cell death (apoptosis)
• Effect is expansion of the neoplastic clone with a
decrease in normal cells
122. Overview of AML cont’
• All acute leukemias begin BEFORE clinical
signs and symptoms occur
• As the tumor volume expands, normal
functional marrow cells decrease
• Characterized by two major features
Ability to proliferate continuously
- Due to mutations affecting growth factors
- Transcription errors
Arrested development of normal cells
THE EXACT CAUSE IS USUALLY UNKNOWN
124. Aetiological factors in AML cont’
2. Environmental factors
• Solvents (benzene)
• Smoking
• Ionizing radiation
Atomic bomb exposure
Nuclear power exposure
• Non-ionizing radiation
• Chemotherapy
Alkylating agents
• Other drugs
Chloramphenicol
Phenylbutazone
3. Viral infections
e.g. HTLV-1
4. Immunological
factors e.g.
hypogammaglobuli
nemia
5. Unknown
– arise de novo in
the majority of
cases
125. Aetiological classification
• Classified by the
cellular appearance of
the primary stem cell
– Common myeloid
progenitor (CMP)
• AML or ANLL
– Common
lymphoid
progenitor (CLP)
• ALL
131. Morphology MPO SBB Specific esterase Nonspecific esterase PAS
M0 Acute myeloblastic
leukemia: mimally
differentiated
>30% blasts
No granules
Not present Not present Not present Not present Not present
M1 Acute myeloblastic
leukemia with no
maturation
>30% blasts
Few granules
+/- Auer rods
Present Present Can be Present Not present Not present
M2 Acute myeloblastic
leukemia with
maturation
>30% blasts Granules
common
+ Auer rods
Present Present Can be Present Not present Not present
M3 Acute promyelocytic
leukemia
>30% blasts
Prominent granules
++ Auer rods
Faggot cells
Present Present Present Not present Not present
M4 Acute
myelomonocytic
leukemia
>30% blasts
>20%monocytes
+ Auer rods
Present Present Present Present Not present
M4 eo Acute myelomonocytic
leukemia
With eosinophilia
>30% blasts
>20%monocytes
>5% abn eos
+ Auer rods
Present Present Present Present Not present
M5 Acute monoblastic
leukemia with or
withour maturation
>30% blasts>80%
monocytes
with/without
differentiation
Can be Present Can be Present Can be Present Present Not present
M6 Acute
erythroleukemia
>30% myeloblasts
>50% megaloblasts
+ Auer rods
Present:
Myeloblasts
Present:
Myeloblasts
Present:
Myeloblasts
Not Present Present:
Erythroblasts
M7 Acute megakaryocytic
leukemia
>30%
Megakaryoblasts
Cytoplasmic budding
Not present Not present Can be Present Not present Not present
132.
133. M1: AML without maturation
• Myeloblast
with Auer rod
• High N:C
ratio
• Fine
chromatin
• Prominent
nuclei
134. M2: AML with maturation
All stages of
neutrophil
maturation
>20%
myeloblasts
Auer rods
common
138. M6: Acute erythroid leukemia
High
number of
RBC
precursors
>20
Myeloblasts
139. M7: Acute Megakaryoblastic
Leukemia
• Peripheral blood
– May see micromegakaryoblasts
– Megakaryocyte fragments
– Cytopenias
– Dysplastic segmented neutrophils and
platelets
• Bone marrow
– Often get “dry tap”
– Fibrosis
140. Prognosis and therapy
• Death often occurs from infection and
hemorrhage in weeks to months unless
therapy is started
• Chemotherapy
– Reduces tumor load
• Bone marrow transplants
142. Overview of ALL
• Causes a wide spectrum of syndromes
– From involvement of bone marrow and peripheral
blood(leukemias) to those that cause masses
(lymphomas) in lymphoid organs.
• The cell of origin may involve the common
lymphoid progenitor cell OR differentiated
progenitors of the T, B or NK cells
– T cell malignancies more aggressive, often involve
extranodal/extramedullary sites like skin, CNS,
mediastinum
– Neoplastic B cells secrete monoclonal proteins(
IgM,IgG, IgA) inappropriately causing increased
viscosity of blood, impairing blood flow
143. Aetiological factors
• Haematologic disorders characterized by
malignant neoplastic proliferation and
accumulation of immature and dysfunctional
hematopoietic cells in the bone marrow.
• The abnormality is an acquired genetic mutation
within a lymphoid precursor cell that gives rise to
a clone of malignant lymphocytes
• These “cells” proliferate in an unregulated manner
and do not develop into mature cells
• Additionally, they have an enhanced ability to
self-renew and have a resistance to cell death.
144. Clinical findings
• Seen in young children
– Peak incidence is between 2-5 years old
• Onset of symptoms can be insidious or abrupt
– Prognosis with treatment good, if no treatment
received, survival very short
• Another smaller peak often seen in the 60’s
– Onset symptoms rapid, fatigue, infections,
bruising
– Prognosis is not good
145. Laboratory features
• WBC count variable from 0.1 - >50 x 109/L
– Usually around 10-12 x 109/L
– Neutropenia
– Lymphoblasts
– Normocytic, normochromic anemia
– Thrombocytopenia (48 - 52 x 109/L)
148. Differentiation between AML and
ALL
• Age
– AML - mainly in adults
– ALL - common in children
• Blood
– AML - anemia, neutropenia, thrombocytopenia, myeloblasts and
promyelocytes
– ALL - anemia, neutropenia, thrombocytopenia, lymphoblasts
and prolymphocytes
• Morphology
– AML - blasts are medium to large with more cytoplasm which
may contain granules, Auer rods, fine nuclear chromatin,
distinct nucleoli
– ALL - blasts are small to medium with scarce cytoplasm, no
granules, fine nuclear chromatin and indistinct nucleoli
• Cytochemistry
– AML - positive peroxidase and Sudan black, negative TdT
– ALL - negative peroxidase and Sudan black, positive TdT
149.
150. FAB classification – L1
Small, uniform
lymphoblasts
Scant cytoplasm,
indistinct nucleoli,
occassional clefting
of nucleus,
chromatin is
clumped
Affects primarily
children
152. FAB classification – L3
L3: Burkitt’s type
Uniform population
of large
lymphoblasts with
deeply basophilic
cytoplasm, vacuoles,
round to oval nuclei
without indentation
Affects adults and
children
153. ALL - prognostic factors
The following are poor prognostic factors in ALL:
• Age <1 year and >10 years
• Male sex
• CNS disease at presentation
• High white cell count
• t(9;22)
• t(4;11)
• Hypodiploidy
154. Acute leukaemias - therapy
– There are four general types of therapy
• Chemotherapy – usually a combination of
drugs is used
• Bone marrow transplant
• Radiotherapy
• Immunotherapy – stimulate the patients
own immune system to mount a response
against the malignant cells
- Monoclonal antibodies –
examples include Rituxin
156. CML
• Also known as Chronic Granulocytic Leukemia (CGL)
• A clonal myeloproliferative disorder of hematopoietic
pluripotent cell transformation characterized by marked
leukocytosis and excessive production of granulocytes at all
stages of maturation
• Etiology unknown (95% of cases)
• Associated with acquired chromosomal abnormality called the
Philadelphia Chromosome
– 90-95% of patients with CML carry Philadelphia Chromosome
– Translocation of chromosomes 9 and 22; t(9:22)
157. Philadelphia Chromosome
Main portion of the long arm of chromosome
22 is deleted and translocated to distal end of
long arm of chromosome 9, and a small part of
chromosome 9 reciprocally translocates to the
broken end of chromosome 22
158.
159. Pathogenesis
Hematopoietic abnormality
- Expansion of granulocytic progenitors and a
decreased sensitivity of the progenitors to
regulation – increased white cell count
- Megakaryocytopoiesis is often expanded
- Erythropoiesis is usually deficient
- Function of the neutrophils and platelet is
nearly normal
160. Pathogenesis
Genetic abnormality
- CML is the result of an acquired genetic abnormality
- A translocation between chromosome 9 and 22 [t(9;22)]
– the Philadelphia chromosome
- The oncogene BCR-ABL encodes an enzyme – tyrosine
phosphokinase
- The function of the normal abl gene product is not
completely understood but it is known to have tyrosine
kinase activity and may play a role in the regulation of
several different growth factor receptors, including those
for epidermal growth factor, platelet derived growth
factor, and colony stimulating factor receptors.
161.
162. Clinical features
• Increasing splenomegally, which is associated
with discomfort, pain or indigestion.
• Refractory anaemia that includes pallor,
weakness and tachychardia.
• Bruising, epistaxis due to abnormal platelet
function.
• Gout or renal impairment due to hyperuricemia.
• Visual disturbances.
• Increased requirements for chemotherapy to
maintain remission.
163. Three Phases of CML
• Chronic
– Controllable with chemotherapy
– Lasts 2-5 years
• Accelerated
– Lasts 6-18 months
– 10-19% blasts in PB and BM
– Low Platelet counts
– Increasing WBC counts
• Blast crisis
– Unresponsive to treatment
– Prognosis less than 6 months
– > 20% blasts in bone marrow
164. Investigation of CML
Laboratory Findings in CML
• Extreme leukocytosis (WBC > 100,000 x 109/L)
• Marked left shift
– Predominance of segmented neutrophils and myelocytes
• Thrombocytosis (can exceed 1000 x 109/L)
– Variant platelet shapes
– Function can be abnormal
• Normochromic-normocytic anemia (Hb 9-13 g/dL)
• Normocytic RBC’s
• Bone marrow: hypercellular with a M:E ratio = 10:1
• Low LAP score (ddx: leukemoid reaction has high LAP)
• Megaloblastic erythropoieis may occur
• Serum uric acid is usually increased.
168. CLL – Terminology
• Chronic – mature cells, signs and symptoms
develop gradually, and often discovered
accidentally; may not be diagnosed for 10-15
years!
• Lymphocytic – lymphoproliferative disorder
• Leukemia – replacement of normal bone marrow
with neoplastic cells, causing anemia,
thrombocytopenia and neutropenia
169. Introduction
• CLL is the most common of the chronic
lymphoid leukemias.
• It is characterised by the accumulation of non-
proliferation mature-appearing lymphocytes
(lymphocytosis) in the blood, marrow, lymph
nodes, and spleen.
170. Introduction cont’
• In most cases, the cells are monoclonal B
lymphocytes that are CD5+
• T cell CLL can occur rarely
• Peak incidence is between 60 - 80years.
• It is the most common form of leukemia in North
America and Europe, but is extremely rare in the
Oriental population
• Affects men twice as often as women
• Incidence rate: 300 cases / 100,000 population
annually.
171. Leukaemogenesis
• Genetic factors have been postulated to play a
role in the high incidence of CLL in some
families
• Cytogenetics
– clonal chromosomal abnormalities are detected in
approximately 50% of CLL patients
– the most common clonal abnormalities are:
• trisomy 12
• structural abnormalities of chromosomes 13, 14 and 11
– patients with abnormal karyotypes have a worse
prognosis
172. Investigation of CLL
• Complete Blood Count (CBC)
– Total WBC count:
– Differential count: lymphocytosis, the absolute
lymphocyte count is >5x109/l and may be up to
300x109/l or more
.
Anemia: normocytic normochromic anemia is
present in later stages, autoimmune haemolysis.
Platelets : thrombocytepenia may occur.
173. Blood and Bone Marrow
Blood film:
70-99% of white cells are mature lymphocytes.
– Lymphocytes are small to slightly larger than normal
with a mature appearance
• Nucleus is round, with block-type chromatin
• Cytoplasm scarce
– Smudge cells (bare nuclei) are common
• Occur due to the cell’s fragility in making a smear
– Prolymphocyte < 10%
Bone marrow aspiration:
Lymphocytic replacement of normal marrow.
178. Cytogenetic studies
The four most common abnormalities are:
• deletion of 13q14,
• trisomy 12,
• deletion of11q23, and
• structural abnormality of 17p involving the
p53 gene.
179. Other haematological findings
• 10 - 25% of patients with CLL develop
autoimmune hemolytic anemia, with a
positive direct antiglobulin test (DAT)
• The marrow aspirates shows greater than 30%
of the nucleated cells as being lymphoid
180. Differential diagnosis
One must distinguish between CLL and
– Prolymphocytic leukemia,
– Hairy cell leukemia,
– Large granular lymphocyte leukemia,
– Sezary syndrome, and
– Circulating lymphoma cells e.g in NHL
• Prolymphocyte leukemia
– This is an aggressive leukemic disorder of mature B or T cells
– > 55% of the lymphocytes are prolymphocytes which are large
with moderate amounts of pale basophilic cytoplasm, mature
condensed chromatin, and a single prominent nucleolus
181. Clinical features of CLL
– Occurs in persons >50 years old
– Men are affected more than women 2:1
– Chronic fatigue, infection
• Result of bone marrow replacement of normal cells
with lymphocytes.
– Skin and organ infiltration and enlargement
– Median survival is 4-5 years, with 30% of patients
surviving 10 years
182. Treatment
– Usually treatment is not required until lymphocytosis causes
other cells to be crowded out resulting in infections.
– Treatment depends on the stage at which the disease is
diagnosed and is usually for the symptoms, not the disease.
• Radiation of localized infiltration
• Chemotherapy given according to stage of disease
• IV gamma globulin for prevention of infection
• Bone marrow transplant done on aggressive cases
183. Prognosis
• 50% of the patients will receive partial
remission.
• < 30% of the patients will got complete
remission.
• 30% of the cases will transfer into PLL.
• 5% of the cases will have Richters syndrome
in which the blastic phase of CLL in lymph
nodes.
185. Definition
• These are a heterogeneous group of diseases
that arise from an overproliferation of
abnormal cells of the lymphoid tissue
(lymphocytes, histiocytes and reticulum cells).
• The spilling of these cells into the peripheral
blood results in a leukemic phase of the
disease.
186. Hodgkin’s disease and NHL
Parameter Hodgkin Lymphoma Non-Hodgkin Lymphoma
Stage Localized Widespread
Distribution Central nodes Peripheral nodes
Mode of spread Close Not close
Extranodal disease Uncommon Common
Peripheral blood Never involved Can be involved
Cell type Abnormal bizarre cells Resembles normal lymphoid cells
188. Background
• First described in 1832 by Dr. Thomas Hodgkin
• Neoplasm of B lymphocytes – large
pleomorphic prominent nucleolus in a halo -
Hodgkin cells
• Reed-Sternberg cell – binucleate Hodgkin cell
with owl eye appearance is pathognomonic
189. What is Hodgkin’s lymphoma?
• A neoplastic disorder with development of specific
infiltrate containing pathologic Reed-Sternberg cells.
• It usually arises in lymph nodes and spreads to
contiguous groups. Extranodal presentations are rare.
• The disease is associated with a defective cellular
immunity.
Probable cause is Epstein-Barr virus
190. HD – Introduction
• Accounts for ~ 30% of all lymphomas
• Classification: Composed of two different disease entities:
– Classical Hodgkin’s: representing ~ 95% of all HDs.
• Nodular sclerosis – low grade
• Mixed cellularity – admixture
• Lymphocyte rich – classical
• Lymphocyte depleted – high grade
– Lymphocyte-predominant HD (LPHD): making up ~ 5%
of cases
* A common factor of both HD types is that neoplastic cells constitute
only a small minority of the cells in the affected tissue, often
corresponding to < 2% of the total tumor
191. HD – Introduction
• Male: Female 2:1 in kids; in adults almost equal M:F
• Mixed cellularity (MC) Hodgkin’s Disease is more
common at younger ages
• More common in immune deficiency patients
192. HD – Introduction cont’
Fatal disease with 90% of untreated patients
dying within 2 to 3 years
With chemotherapy, >80% of patients
suffering from HD are cured.
Pathogenesis of HD is still largely unknown.
HD nearly always arises and disseminates in
lymph nodes
193. HD – Clinical presentation
• Non-tender lymph node enlargement (localized)
– neck and supraclavicular area
– mediastinal adenopathy
– other (abdominal, extranodal disease)
• Systemic symptoms (B symptoms)
– fever
– night sweats
– unexplained weight loss (10% per 6 months)
• Other symptoms
– fatigue, weakness, pruritus
– cough , chest pain, shortness of breath, vena cava syndrome
– abdominal pain, bowel disturbances, ascites
– bone pain
194. HD – Clinical presentation
SIGNS & SYMPTOMS % OF PATIENTS
Lymphadenopathy 90
Mediastinal mass 60
“B” symptoms 30
Fever, weight loss, night sweats
Hepatosplenomegaly 25
• Most commonly involved lymph nodes are the
cervical and supraclavicular in 75% of cases
• Bone marrow is involved in 5% of patients
195. HD – Pathology
– Characteristic cell is the Reed-Sternberg
• Giant size (up to 45µm in diameter)
• Abundant acidophilic cytoplasm
• Multinucleated
• Gigantic nucleoli
196. HD – Diagnosis
• The Reed-Sternberg cell is pathognomonic
• Excisional biopsy of a lymph node.
Fine needle aspirate is not sufficient to make the
diagnosis of Hodgkin’s disease
197.
198. Classical HD
• Nodular sclerosing
Is the most common subtype and is composed of large
tumor nodules showing scattered classical RS cells set in a
background of reactive lymphocytes, eosinophils and
plasma cells with varying degrees of collagen
fibrosis/sclerosis.
• Mixed cellularity
Is a common subtype and is composed of numerous classic
RS cells admixed with numerous inflammatory cells
including lymphocytes, histiocytes, eosinophils, and plasma
cells without sclerosis. This type is most often associated
with EBV infection.
199. HD – Staging
– Stage I - single lymph node region or single organ
– Stage II - two or more lymph node regions on the same
side of the diaphragm
– Stage III - involvement of lymph nodes on both sides of the
diaphragm
– Stage IV - disseminated into other tissues and organs
including bone marrow
200. HD – Treatment and prognosis
– Radiation of localized involvement
– Chemotherapy
– Combination of above
– With early diagnosis, long-term disease-free
survival is seen in about 75% of cases.
202. Background
• Non-Hodgkin’s lymphomas (NHL) are a
heterogeneous group of lymphomas: there are
many different subtypes, every few years the
classification is updated. Today, morphology,
immunophenotype, molecular, cytogenetics, and
other techniques are used for diagnosis.
• Treatment generally depends on the
aggressiveness of the disease (indolent,
aggressive, or very aggressive)
203. NHL
• Cause is unknown. Predisposing factors seem to be
chemicals, ionizing radiation and certain viruses. Reed-
Sternberg cells are NOT present.
• The types of non- Hodgkin’s lymphoma reflect the
developmental stages of lymphocytes.
• Each type of lymphoma can be viewed as a lymphocyte
arrested at a certain stage of development and
transformed into a malignant cell.
• 85% B cell origin, the rest T or null cell.
204. NHL – Aetiology
• Immune suppression
– congenital (Wiskott-Aldrich)
– organ transplant (cyclosporine)
– AIDS
– increasing age
• DNA repair defects
– ataxia telangiectasia
– xeroderma pigmentosum
205. NHL – Aetiology cont’
• Chronic inflammation and antigenic stimulation
– Helicobacter pylori inflammation, stomach
– Chlamydia psittaci inflammation, ocular adnexal
tissues
– Sjögren’s syndrome
• Viral causes
– EBV and Burkitt’s lymphoma
– HTLV-I and T cell leukemia-lymphoma
– HTLV-V and cutaneous T cell lymphoma
– Hepatitis C
206. NHL Behaviour
• Indolent – these lymphomas grow slowly. The
majority of NHLs are considered indolent. Indolent
lymphomas are generally considered incurable with
chemotherapy and/or radiation therapy.
• Aggressive – these lymphomas have a rapid growth
pattern. This is the second most common form of NHL
and are curable with chemotherapy.
• Very Aggressive – these lymphomas grow very rapidly.
They account for a small proportion of NHLs and can be
treated with chemotherapy. Unless treated rapidly,
these lymphomas can be life threatening.
207. WHO/REAL Classification of lymphoid
neoplasms
B-Cell Neoplasms
Precursor B-cell neoplasm
Precursor B-lymphoblastic leukemia/lymphoma
(precursor B-acute lymphoblastic leukemia)
Mature (peripheral) B-neoplasms
B-cell chronic lymphocytic leukemia / small lymphocytic
lymphoma
B-cell prolymphocytic leukemia
Lymphoplasmacytic lymphoma‡
Splenic marginal zone B-cell lymphoma
(+ villous lymphocytes)*
Hairy cell leukemia
Plasma cell myeloma/plasmacytoma
Extranodal marginal zone B-cell lymphoma of MALT type
Nodal marginal zone B-cell lymphoma
(+ monocytoid B cells)*
Follicular lymphoma
Mantle cell lymphoma
Diffuse large B-cell lymphoma
Mediastinal large B-cell lymphoma
Primary effusion lymphoma†
Burkitt’s lymphoma/Burkitt cell leukemia§
T and NK-Cell Neoplasms
Precursor T-cell neoplasm
Precursor T-lymphoblastic leukemia/lymphoma
Mature (peripheral) T neoplasms
T-cell chronic lymphocytic leukemia / small
lymphocytic lymphoma
T-cell prolymphocytic leukemia
T-cell granular lymphocytic leukemiaII
Aggressive NK leukemia
Adult T-cell lymphoma/leukemia (HTLV-1+)
Extranodal NK/T-cell lymphoma, nasal type#
Enteropathy-like T-cell lymphoma**
Hepatosplenic γδ T-cell lymphoma*
Subcutaneous panniculitis-like T-cell lymphoma*
Mycosis fungoides/Sézary syndrome
Anaplastic large cell lymphoma, T/null cell,
primary cutaneous type
Peripheral T-cell lymphoma, not otherwise characterized
Angioimmunoblastic T-cell lymphoma
Anaplastic large cell lymphoma, T/null cell,
primary systemic type
Hodgkin’s Lymphoma (Hodgkin’s Disease)
Nodular lymphocyte predominance Hodgkin’s lymphoma
Classic Hodgkin’s lymphoma
Nodular sclerosis Hodgkin’s lymphoma (grades 1 and 2)
Lymphocyte-rich classic Hodgkin’s lymphoma
Mixed cellularity Hodgkin’s lymphoma
Lymphocyte depletion Hodgkin’s lymphoma
† Not described in REAL classification
§ Includes the so-called Burkitt-like lymphomas
** Formerly known as intestinal T-cell lymphoma
# Formerly know as angiocentric lymphoma
208. Epidemiology of NHL
• Indolent lymphomas are rare in young people
and increase in incidence with age.
• Large cell lymphoma (DHL) is less age related,
and is among most common cancers affecting
the young.
• Burkitt’s and lymphoblastic lymphoma are
common in adolescents.
• AIDS patients develop aggressive, high grade
lymphomas.
209. NHL – Clinical features
• Lymphadenopathy
• Cytopenias
• Systemic symptoms
• Hepatosplenomegaly
• Fever
• Night sweats
210. NHL – Diagnosis
• Excisional biopsy is preferred to show nodal architecture (follicular vs
diffuse).
• Immunohistochemistry to confirm cells are lymphoid
– LCA (leukocyte common antigen)
– Monoclonal staining with Ig or Ig
• Flow cytometry:
– CD 19, CD20 for B cell lymphomas
– CD 3, CD 4, CD8 for T cell lymphomas
• Chromosome changes
– 14;18 translocation in follicular lymphoma
• bcl-2 oncogene
– t(8;14), t(2;8), t(8;22) in Burkitt’s lymphoma
• c-myc oncogene
211. Burkitt’s lymphoma (BL)
– Endemic in Africa
– 1/3 of all non-African paediatric lymphomas
– Found in immunocompromised patients, particularly AIDS
patients
– Tumor growth rate is the highest of any tumor with growth
doubling each day.
– Rapid growth and tumor cell death results in “starry sky”
appearance of the biopsy caused by macrophages cleaning
up the dead cells.
– Characteristic overgrowth of facial bones in the African
variety and abdominal mass in the non-endemic variety.
– Cytogenetic translocation t(8:14)
214. NHL – Lymphoblastic type
• Very aggressive
• Treatment is with acute lymphocytic leukemia
regimen
• Often requires high-dose therapy and
allogeneic transplantation for
relapsed/refractory disease
215. Working formulation
• Low Grade
– Small Lymphocytic
– Follicular small-cleaved cell
– Follicular mixed small-cleaved and large cell
• Intermediate Grade
– Follicular large cell
– Diffuse small cleaved cell
– Diffuse mixed small and large cell
– Diffuse large cell
• High Grade
– Large cell immunoblastic
– Lymphoblastic
– Small non-cleaved cell (Burkitt's and non-Burkitt's type)
217. Difinition
• Multiple myeloma (MM) is characterized by the
neoplastic proliferation of a single clone of plasma
cells producing a monoclonal immunoglobulin (Ig).
• Paraproteinaemia refers to the presence of a
monoclonal Ig band in serum. Normally serum Igs are
polyclonal and represent the combined output of
millions of different plasma cells.
• In MM, there is plasma cell accumulation in the BM;
monoclonal protein in serum and/or urine; and
related tissue damage.
218. Incidence
• MM occurs in all races and all geographic
locations
• African Americans and blacks from Africa
have a 2 - 3 times the risk in whites
• The risk is lower in Asians from Japan and
in Mexicans
• Slightly more frequent in men than in
women (1.4:1)
219. Age
• MM is a disease of the elderly
• The median age at diagnosis is 66 years
• Only 10 percent of patients are younger than
50 years
• Only 2 percent of patients are younger than
40 years
220. Epidemiology
• Family History = 4 fold increased risk (autosomal dominant)
• Associated conditions: Obesity, RA, Monoclonal
gammopathy of undetermined significance (MGUS)
• Occupational Exposures:
o Ionizing radiation
o Farming pesticides
o Petroleum Workers
• MGUS
o Premalignant condition
o Abnormal production of monoclonal Ig by plasma cell
o This also happens in MM, however:
Fewer M-proteins
No End Organ Damage
o 2 percent population > 50 years
o Risk of progressing to MM > 1 percent each year.
221. Pathophysiology
• Normally, plasma cells produce
immunoglobulins to fight infection
• However, in MM and MGUS a single
cloned plasma cell proliferates and
overproduces the same Ig (i.e. the "M-
protein" or "paraprotein" )
o The M-protein is usually an IgG
• MM cells can also just produce the light
chain component (instead of the entire Ig)
224. Pathophysiology cont’
• Consequence of producing lots of
monoclonal Ig:
o Hyperviscosity
o Kidney damage (from light chains only)
o Bone pain, hypercalcemia and pathologic
fractures from bone lesions.
o Anaemia/Pancytopenia from bone
marrow invasion
225. Pathophysiology cont’
• 80% of cases of MM arise de novo
• 20% percent follow MGUS
• Risk factors for progression from MGUS to
MM include:
o An elevated M protein level > 1.5 g/dl
o A non-IgG MGUS
• Patients with MGUS should be monitored
closely for 6 to 12 months.
226. Clinical Presentation
• Many patients (58%) initially present with
unexplained backache or bone pain.
o Long bones, ribs, skull, and pelvis are commonly
involved, and most patients have multiple lytic
skeletal lesions.
o Pathologic fracture is the presenting symptom in
26 to 34 %
• Anemia (73%), Elevated BUN (48%),
Fatigue/weakness(33%), Hypercalcemia (28%),
Weight loss (25%)
• Other: Recurrent infections, Headaches/visual
changes, parenthesizes (5%), fever, organomegaly
(1%).
227. Clinical Presentation cont’
• 34 percent of patients are asymptomatic
at presentation with incidental findings
o Increased total protein,
o Renal inefficiency, CR > 2 mg/dl
o Hypercalcemia > 11 mg/dl
o Anemia, Hb < 10 g/dl
• More asymptomatic patients are being
identified with increase in "routine blood
work-up"
228. Rare presentations
• Soft tissue or solitary bone
masses (plasmacytomas)
• Hyperviscosity-induced arterial
infarctions or venous thrombosis
• Concomitant amyloidosis with
gastrointestinal symptoms,
peripheral neuropathy, or
cardiomegaly
229. Helpful Mnemonic: M-CRAB
• M – Monoclonal protein
• C – Calcium
• R – Renal failure
• A – Anemia
• B – Bone pain with lytic lesions
230. Diagnosis - Tests
• SPEP and UPEP with immunofixation
o 80% patients have +ve SPEP
o The remainder have +ve UPEP only
o About 3% have "non secretory" MM ( neg SPEP &
UPEP)
• Urine dipstick tests
o Classic test for Bence-Jones proteins (free light chains),
but insensitive
• Bone marrow
o Anyone with abnormal SPEP/UPEP
o May required multiple aspirates for focal dyscrasias
• CBC, peripheral smear (rouleaux), BUN/CR, Ca, ESR/CRP
231.
232. Imaging work-up
• Skeletal Survey
o Skull, spine, long bones,
ribs, pelvis
• MRI
o More sensitive
o But, generally reserved for
suspected spinal lesions
233. Diagnosis
• Depends on 3 principal findings
o Monoclonal protein in serum and/or urine
o Increased plasma cells in the BM
o Related tissue or organ impairment
e.g. Bone disease, Renal impairment, Anemia,
Hypercalcemia, Hyperviscosity, Amyloidosis or
Recurrent infection
234. Diagnostic criteria
• Major criteria
1. Plasmacytoma on tissue biopsy
2. BM infiltration with >30% plasma cells
3. Monoclonal globulin spike on SPEP (IgG >35 g/l; IgA >20 g/l) or
UPEP (>1 g/24hr of κ or λ light chains)
• Minor criteria
(a) BM infiltration with 10-30% plasma cells
(b) Paraprotein less than in 3 above
(c) Lytic bone lesions
(d) Normal IgM <0.5 g/l; IgA <1 g/l or IgG <6 g/l
Diagnosis requires a minimum of 2 Major criteria OR 1 Major
+ 1 Minor criterion OR 3 Minor criteria including (a) and (b).
236. Treatment - Symptomatic
Disease
• Autologous stem cell transplantation (ASCT) is
treatment of choice
o Older patients > 65 may not physically tolerate
the procedure
o ASCT preceeded by chemotherapy induction
protocols (vincristine, doxorubicin,
dexamethasone)
o 5 year survival 35%
237. Managing compliations of MM
• Renal Insufficiency
o Identifying reversible causes. Dialysis PRN.
o Plasmaphereis for hyperviscosity induced
thrombosis
• Anemia
o Erythopoetin, Blood transfusion
• Infection
o Treat aggressively with broad spectrum
antibiotics
238. Summary
• Consider MM in elderly patients with new
onset of bone pain/fatigue/CRAB symptoms
not resolving with treatment
• Basic work up: SPEP/UPEP (+/-
immunofixation), CBC, peripheral smear,
BUN/CR, Ca, skeletal survey
• DDx: MGUS, asymptomatic vs symptomatic,
Waldenstrom’s, Amyloidosis, NHL.
• Manage Complications: Bisphosphonates,
Orthopedic referral PRN, CRAB.
