Leukocyte Disorder PART 2

Leukemia Classification

Acute lymphoytie leukemia Acute myslaid leukemia

Chronic lymphocytic leukemia Chronic myeloid leukermia
(CLL)

Acute Unregulated proliferation of the lymphoid stem

Lymphoproliferative cells
Disorders
Classified morphologically using FAB criteria, or
immunologically using CD markers to determine
cell lineage (T or B cel).

Clinical symptoms: fever, bone/joint pain,

bleeding, hepatospenomegaly.

Laboratory: Neutropenia, anemia, and

thrombocytopenia; variable WBC count,
hypercellular marrow with bone marrow blasts
220% (WHO) or >30% (FAB).

oLymphoblasts stain PAS positive; Sudan Black B

and myeloperoxidase negative.

DFAB classification of acute lymphoblastic

leukemia (ALL)
Acute leukemias-L1
Li- This is the most
common form found in
children and it has the best 00
prognosis.
The cell size is small with
fine or clumped homogenous
nuclear chromatin and
absent or indistinct nucleoli. O 00
The nuclear shape is regular,
occasionally clefting or
indented.
The cytoplasm is scant, with
slight to moderate basophilia
and variable vacuoles.

ALL
peripheral blood smear

normocytic, normochromic anemia

thrombocytopenia
WBC may be low, normal, or high

Most T cell ALLs are FAB 1

peripheral B-cell

phosphatese PAS+
Most common in children
Arises from lymphoid tissue
About 75h% are nul cellype.
20-25 are I cell ypeand
few are B cell type.
Bimodal distrbutioo
Malefemale-21

ALL-in contrast to the myeloblast, the

lymphoblast is a small blast with scant cytoplasm
dense chromatin, indistinct nucleoli, and no auer
rods

Acute leukemias-L2
L2-This is the most
frequent ALL found in
adults.
The cell size is large and 0 00
heterogenous with variable
nuclear chromatin and
prominent nucleoli.
.The nucleus is irregular,
clefting and indented.
The cytoplasm is variable
and often moderate to
abundant with variable
basophilia and variable
vacuoles. 0

Acute leukemias-L3 (BURKITT'S

LEUKEMIA)
L3-This is the rarest
form of ALL.
The cell size is large, with
fine, homogenous nuclear
chromatin containing
prominent nucleoli.
The nucleus is regular oval
to round.
.The cytoplasm is
moderately abundant and
is deeply basophilic and
vacuolated.

Acute ALL-L3
Lymphoproliferative
Disorders Blasts are deeply
basophilic
Vacuolated cytoplasm
DLeukemic phase of
Burkitt's lymphoma
Seen in both adults and
children
Poor prognosis
ALL FAB L3 are of B cell
Starry sky lineage

High grade from non

Hodgkin lympoma phase ol
FAB L3 leukemia.

Endemic in East Africa with

association with Epstein-
Barr virus; children present
with jaw/facial bone
tumors.
DU.S.variant seen in
children and young adults;
present with abdominal
mass.

Immunophenotyping of ALL
CD marker characteristics of B cell lineage
D Expressed by specific cell lines at different maturation
stages; as cell matures, losses some antigens and
expresses new ones.

CProgenitor B cells are CD19, CD34, Tdt (terminal

deoxynucleotidyl transferase) positive; CD10 (CALLA)
negative. This is the least mature B cell.

Early pre-B cells ALL are CD10 (CALLA), CD 19, CD34

and Tdt positive. This is the most common.

CD marker characteristics of B cell lineage

Pre-B cells ALL are CD10 (CALLA), CD19, CD20, Tdt
positive. This is the second most common subtype.

B cells ALL early B are CD19 and CD20 positive; Tdt

negative. This is the most mature B cell and least
common subtype.

CD19 is the only marker expressed through all stages

of B cells
CD marker characteristics of T cell lineage
Differentiated from B cells using markers present on all
T cells including CD2, CD3, CD5 and CD7 (pan T cell
markers). Immature T cell translocations are Tdt
positive.

Immature T cells can have both or neither CD4 and

CD8. Mature T cells have one or the other, but not both.

Tcell ALL occurs most often in males; mediastinal

mass is a common finding.

Genetic translocations are helpful in diagnosis. Common

ones include

FAB L3/Burkitt's lymphoma-t(8,14) with

rearrangement of MYC oncogene.

Pre-B cell ALL, associated with t(9;22)

DB cell ALL, associated with t(4:11)

Tcell ALL associated with t(7;11)

chronic lymphoproliferative disorders - chronic lymphocytic

leukemia (CLL)
Found in adults over 60 years old; more common in
males (2:1); survival rate of 5-10 years.
B cell malignancy (CD19, CD20 positive)

DOften asymptomatic and diagnosed secondary to

other conditions.

Laboratory: Bone marrow hypercellular; blood shows

absolute lymphocytosis of > 5.0 x 10/;
homogeneous, small, hyperclumped Ilymphocytes and
smudge cells.

Anemia is not usually present unless secondary to

warm autoimmune hemolytic anemia (frequent
complication).

Small lymphocyte lymphoma (SLL) is the lymphoma

phase of CLL

Chronic Lymphocytic Leukemia (CLL)

Indolent clinical course

median survival:4-6 yrs
occasional transformation to large non-Hodgkin's lymphoma
(Richters
syndrome)-3 to 5 %

Hairy Cell Leukemia (reticuloendotheliosis)

clinical

M>F (3-5:1)
splenic red pulp involvement-> red pulp "lake
tends to follow an indolent course
pancytopenia - most prominent feature
granulocytopenia recurent bacterial infection
anemia fatigue
- thrombocytopenia > bleeding
good response to some chemotherapy regimen
Hairy Cell Leukemia

uncommon variant of peripheral B-cell neoplasm

clinically middle age to elderly (younger than CLL)
Splenic red pulp involvement
histologically lymphocyte with finger-like projections
phenotypically TRAP (Tartrate Resistant Acid Phosphatase
CD19, CD20

Prolymphocytic leukemia (PLL

Found in adults; more common in males

Marked splenomegaly

T CELL PROLYMPHOCYTIC LEUKEMIA

B CELL PROLYMPHOCYTIC LEUKEMIA
MOST COMMON

Laboratory:

Characterized by lymphocytosis (>100 x 109/L)

with many prolymphocytes; anemia and
thrombocytopenia

Both B and T cell types are aggressive and

respond p0orly to treatment

other lymphoid malignancy

Plasma cell neoplasms:
Multiple myeloma

Monoclonal gammopathy causes B cell

production of excessive IgG (most common)
or IgA, with decreased production of the
other immunoglobulins.

Found in adults over 60 years old; incidence

higher in males

OMultiple skeletal system tumors of plasma

cells (myeloma cells) cause lytic bone
lesions and hypercalcemia.

Multiple myeloma

CIdentified on serum protein electrophoresis by an

"M" spike in the gamma-globulin region
immunoglobulin class determined using
immunoelectrophoresis and quantified using an
immunoassay method.

Excessive IgG or IgA production by myeloma cells

causes increased blood viscosity.

Abnormal Ig binds to platelets, blocking receptor

sites for coagulation factor binding--- results in
prolonged bleeding.

0Laboratory: Bone marrow plasma cells >30%,

marked rouleaux, increased ESR rate, blue
background to blood smear, plasma cells and
lymphocytes on blood smear.

Bence Jones proteins (free light chains

kappa or lambda) found in the urine; toxic to
tubular epithelial cells; cause kidney damage
Pathology: expanding plasma cell mass,
overproduction of monoclonal Igs, production
of osteoclast activating factor & other
cytokines

Waldenstrom macroglobulinemia (malignancy

of the lymphoplasmacytoid cells)

Monoclonal gammopathy causes B cell

production of excessive lgM (macroglobulin)
and decreased production of the other Igs.

CFound in adults over 60 years old.

Lymphadenopathy and
hepatosplenomegaly; no bone tumors.

Excessive lgM production causes increased

blood viscosity.

ldentified on serum protein electrophoresis by

an "M" spike in the gamma globulin region; Ig
class determined using immuno
electrophoresis and quantified using an
immunoassay method.

Abnormal lg may interfere with platelet

function, fibrin polymerization and the function
of other coagulation proteins
Results from characterization studies of
urinary lgs indicate that light chains (Bence
Jones protein) usually of the kappa type are
found in the urine.

Laboratory: Marked rouleaux, increased ESR,

blue background to blood smear,
plasmacytoid lymphocytes, plasma cells and
lymphocytes on blood smear.

Lymphoma

Proliferation of malignant cells in solid

lymphatic tissue.

Initially localized; may spread to bone

marrow and blood.

Clinical symptoms: Lymphadenopathy

Diagnosis: Tissue biopsy, CD surface

markers, cytogenetics, DNA analysis/PCR,
peripheral blood examination & CBC

WHO groups the lymphomas into Hodgkin,B

cell, and T/NK cell (non-Hodgkin) neoplasms

D Hodgkin lymphoma (classical)

40% lymphomas; seen in patients

between15 and 35 years of age and
over 55 years of age; seen more
frequently in males; certain subtypes
have an EBV association
Hodgkin lymphoma (classical)

Reed-Sternberg (RS) cells found in

lymph node biopsy (key diagnostic test)
are large, multinucleated cells each
with prominent, large nucleoli; B cell
lineage

Hodgkin lymphoma subtypes using WHO

classification
a. Nodular sclerosis -70% are this
subtype, lowest EBV association
b. Mixed cellularity- 20% are this
subtype, highest EBV association
c. Lymphocyte rich
d. Lymphocyte depleted - uncommon
e. All subtypes are associated with RS
cells

Laboratory: Mild anemia, eosinophilia, and

monocytosis, increased LAP score and
ESR during active disease

Non-Hodgkin lymphoma

WHO Sseparates B cell and T/NK cell

neoplasms into conditions with precursor
cells or mature cells
60% of lymphomas; seen in patients over
60 years of age
Enlarged lymph nodes or gastrointestinal
(GI) tumors

B cell neoplasms are more common, include

Burkitt (lymphoma phase of Burkitt
lymphoma), mantle cell, follicular and other
lymphomas.

Cells can be small and mature (e.g. small

lymphocytic lymphoma) or large and primitive
(e.g. Precursor B cell lymphoblastic
lymphoma)

D Can be slow growing or very aggressive

Mycosis fungoides (cutaneous T cell

lymphomas)

Classified by WHO as a T/NK cell neoplasm

(non-Hodgkin lymphoma)

OSeen in patients over 50 years of age

Cutaneous lymphoma causes skin itching,

leading to ulcerative tumors

CD2, CD3 and CD4 positive

Mycosis fungoides (cutaneous T cell

lymphomas)
Sezary syndrome, a variant of mycosis
fungoides, presents as a disseminated
disease with widespread skin involvement
and circulating lymphoma cells
CD2, CD3 and CD4 positive

MYELOID
MALIGNANCIES
Leukemia characterized by proliferation of myeloid
tissue (as of the bone marrow and spleen) and an
abnomal increase in the number of granulocytes,
myelocytes, and myeloblasts in the circulating blood

acute myeloproliferative disorders

Unregulated proliferation of the myeloid stem cell;
classified using morphology, cytochemical stains, CD
markers, cytogenetics; WHO classification standard for
diagnosis; FAB classification still widely taught Plasma cell
neoplasms.

Platelets, erythrocytes, granulocytes, and/or

monocytes can be affected

Found mainly in middle-aged adults; also children <1

year old

OClinical symptoms: Fever, malaise, weight loss,

petechiae, bruises, mild hepatosplenomegaly

Laboratory: Neutropenia, anemia, and

thrombocytopenia; variable WBC count,
hypercellular marrow with bone marrow blasts
220% (WHO) or >30% (FAB)
Acute myelogenous leukemia (AML)
(in children -associated with the best prognosis
for a cure)

FAB MO-Undifferentiated
Blasts exhibit myeloid markers CD13, CD33, and CD34 but
stain negatively with the usual cytochemical stains,
myeloperoxidase (MPO), and SBB. Constitutes <5% AMLs.
Blasts are negative for B and T lymphoid antigens, platelet
glycoproteins and erythroid glycoprotein A

FAB M1 (AML without maturation) shows 90% or

more marrow myeloblasts; may have Auer rods
(fused primary granules)
Myeloblast with auer rod AML-M1

Blast cels have few

a2zurophilic granules
May have auer rods

FAB M2 (AML with maturation) shows <90% marrow

myeloblasts; may have Auer rods ; chromosome
abnormality t(8;21)
AML -M2

Multiple granules
0 May have Auer rods
With granulocytic
differentiation

FAB M2 (AML with maturation)

Both FAB M1 and FAB M2 are SBB, MPO and

specific esterase positive
FAB M1 and FAB M2 account for 50% of the
AMLs

oCD13 and CD33 positive (pan myelid markers)

Acute promyelocytic leukemia (APL; FAB M3)

Characterized by >30% marrow promyelocytes

with bundles of Auer rods (faggot cells); heavy
azurophilic granulation
AML- M3

Abnormal
promyelocytes
Prominent granules
Multiple Auer rods

Acute promyelocytic leukemia (APL; FAB M3)

Clinical symptoms: Severe bleedin9

hepatomegaly, DIC (promyelocytes have
procoagulant activity)

Account for 5% of the AMLs

SBB, MP0 and specific esterase positive

CD13 and CD33 positive; diagnostic

chromosome abnormality PML/RARA
oncogene involved t(15, 17)
Clinical symptoms: Severe bleeding, hepatomegaly,
DIC (promyelocytes have procoagulant activity)

DAccount for 5% of the AMLs

SBB, MPO and specific esterase positive

CD13 and CD33 positive; diagnostic chromosome

abnormality t(15;17) PML/RARA oncogene involved

Lymphocytes are also morphologically similar to

Burkitt lymphoma

Acute myelomonocytic leukemia (AMML; FAB M4)

Characterized by 220% (WHO) or >30% (FAB)

marrow myeloblasts with >20% cells of
monocytic crigin; may have Auer rods

Proliferation of unipotential stem cell CFU-GM that

gives rise to both granulocytes and monocytes

Accounts for 30% of the AMLs

Increased serum/urine lysozymes

SBB, MPO, and specific and nonspecific esterase

positive
CD13 and CD33 positive (myeloid) and CD14 positive
(monocytes)

MAXo is a subclass of AMML that presents with

eosinophilia

AML- M4

Have monocytoid
diferentiation
20% or more is
monocytoid cels

AML-MA

Note monoblasts and promonocytes

M4-acute
myelomonoblastic
eukemia
Both myeloblasts and
monoblasts are seen in
the bone marowand
penpheral blood
Infitradon of
extramedulary sitesis
more common than
with the pure
granulocytic variants

Acute monocytic leukemia (AMoL; FAB M5)

Characterized by 220% (WHO) or >30% (FAB)

marrow monoblasts
Accounts for 10% of the AMLs
Nonspecific esterase positive; CD14 positive

Called the true monocytic leukemia and follows an

acute or subacute course characterized by monoblasts,
promonocytes, and monocytes

Acute monocytic leukemia (AMoL; FAB M5)

Contains two variants

M5a-seen in children with >80% monoblasts

in the bone marrow
M5b seen in middle-aged adults with <80%
monoblasts in the bone marroW

AML- M5a AML- M5b

Monoblastic Monocytic
80% of blasts are 80% of blasts are
monoblasts monoblasts

Acute erythroleukemia (AEL, Di Guglielmo syndrome;

FAB M6)

Characterized by 220% (WHO) or >30% (FAB)

marrow myeloblasts and >50% dysplastic
marrow normoblasts

Accounts for 5% AMLs

Malignant normoblasts are PAS positive. The

myeloblasts are SBB and MPO positive

Malignant normoblasts are CD45 and CD71

(glycophorin A) positive. Myeloblasts are CD13,
CD15 and CD33 positive

Malignant normoblasts are CD45 and CD71

(glycophorin A) positive. Myeloblasts are CD13,
CD15 and CD33 positive

AML-M6

Predominance of
erythroblast
Dyserythropoiesis

Acute megakaryocytic leukem ia (AMegL; FAB M7)

Characterized by a proliferation of
megakaryoblasts and atypical megakaryocytes
in the bone marrow; blasts may have cytoplasmic
blebs

Accounts for <1% of the AMLs

Marrow aspiration results in dry tap, blood shows
pancytopenia

oCD41, CD42, and CD 61 (platelet markers)

positive

AML- M7

Megakaryoblast with
00 cytoplasmic blebs
Bilineage leukemias contain two cell populations. One
population expresses myeloid antigens; the other
population expresses lymphoid antigens

Biphenotypic leukemias occur when myeloid and

lymphoid antigens are expressed on the same cell;
poor prognosis

The WHO classification of acute myeloid leukemias

has more than 20 subtypes; all have 220 marrow
blasts

CHRONIC MYELOID
LEUKEMIA
Aneoplasm of hemopoietic stem cells caused by the
Philadelphia' chromosome t(9:22)
AWO-phase disease

chronic myeloproliferative disorders

Characterized by hypercellular marrow, erythrocytosis,
granulocytosis and thrombocytosis

Defect of the myeloid stem cell

Named for the cell line most greatly affected
O All may terminate in acute leukemia

Molecular diagnostic studies are helpful in identifying

oncogenes

JAK 2 (V617F) oncogene is implicatedin

polycythemia vera (80%6) chronic idiopathic
myelofibrosis (50%) and essential thrombocytopenia
(40%)
The BCR/ABL oncogene is associated with chronic
myelogenous leukemia. Correlates with t(9;22). The
prognosis is better when the abnormality is present

Myelo-proliferative Disoders

Chronic myelogenous leukemia (also called CML or chronic

granulocytic leukemia) presents with proliferation of
granulocytes.

The bone marrow makes too many wbcs.

Found mainly in adults 45 years of age and older, rarely

ocCur in children.

Clinical symptoms: weight loss, splenomegaly, fever, night

sweats and malaise.

Bone marrow has an increased M:E ratio

Laboratory Findings: mild anemia, wbc between 50 and 500

x 109/L, with all stages of granulocytic production (shift to
the left), including early forms of eosinophils and basophils,
Myelocytes predominate, may have a few circulating blasts.

CML cam mimic neutrophilic leukemoid reaction (NLR), LAP

score is, low in CML and high in NLR.

Philadelphia chromosome, t(9, 22), is present in virtually all

patients. All cell lines are affected except lymphocytes.
Few who lack the chromosome have a worse prognosis
Few who lack the chromosome have a worse prognosis.
Chronic phase can last up to 5 years, accelerated phase
(blast crisis) ultimately leads to acute leukemia in most
patients.
Recent therapies are improving the prognosis.

Essential Thrombocythemia (ET) is a chronic

myeloproliferative neoplasm (MPN) characterized by an
increased number of platelets in the blood.

Most commonly diagnosed in women over the age of 50, ET

is associated with a proliferation of platelet precursors in the
bone marrow and complications frequently include blood
clotting and/or bleeding.

Less common consequences in the later stages of ET include

a transformation to myelofibrosis (marrow scarring) or acute
leukemia.

Laboratory Findings:
Platelets commonly greater than 1000 x 10/L or
450 x 10°/L, giant forms, platelet function abnormalities,
leukocytosis
Must be differentiated from reactive thrombocytes
and polycythemia vera.

Polycythemia vera

A malignant hyperplasia of the multipotential myeloid stem

cell causes increase in all cell lines (polycythemia),
erythrocytes most greatly increased despite decreased
erythropoietin, inappropriate erythropoiesis.
High blood viscosity can cause high blood pressure, stroke
and heart attack.

Found in adults 50 years of age and older.

Must differentiate from other forms of polycythemia

Secondary polycythemia
a. Increase in RBC mass is an appropriate response to
increased EPO or tissue hypoxia. Plasma volume, leukocyte
count and platelet count normal.

b. Can be caused by smoking, emphysema or high altitude

Relative (pseudo) polycythemia

a. Decreased plasma volume witha normal RBC mass
caused by dehydration (diarrhea, diuretics or burns)

b. Increased Hgb, normal leukocyte & platelet count,

normal EPO.

Chronic neutrophilic leukemia is another rare

myeloproliferative neoplasms in which too many neutrophils
are made in the bone marrow.

These cells spill out into the circulating blood and tend to
accumulate in the liver and spleen which become enlarged
as a result.

Usually a slow progressing disease closely related to chronic

myeloid leukemia.
MPNs Diagnosis
A sign shared by all myeloproliferative disorders
with the exception of Essential thrombocythemia
(thrombocytosis) is an enlarged spleen

Conduct the following tests

1. Blood tests: To find abnormal types or numbers of red or
white blood cell. They can also detect anemia or leukemia.

2. Bone Marrow Biopsy: similar to blood tests and can also

detect cancers

3. Cytogenic analysis: To find changes in the chromosomes

i.e. Philadelphia.
4. JAK 2 gene mutation test: Look for a change (mutation)
in a piece of genetic material in the cells (a gene) called JAK
2. This is often found in three of the Myeloproliferative
Disorders, PV, ET or prinmary myelofibrosis.

Cytogenetics
cytogenetics studies can now be used for diagnosis
and for prognosis of hematologic malignancies.
Many leukemias (and lymphomas) are characterized by
specific chromosomal abnormalities, including specific
translocations and aneuploidy. The specific type of
malignancy can be identified based on the specific
abnormality or translocation. These may be identified
by
Looking at the karyotypes of the chromsomes from the
abnormal cells
DNA based tests - these tests are very useful for following the
course of the disease
RT-PCR
Southern blotting
A normal karyotype is usually associated with a better
prognosis.

PERIPHERAL SMEAR
LEUKEMOID REACTION CML CNL

LEUKEMOID REACTION
Leucocytes consist mostly of
mature neutrophils.
The differential count discloses a
marked left shift, as
evidenced by the presence of
myelocytes and metamyeocytes
In addition PS discloses toxic
granulation, Doëhle bodies, and
cytoplasmic vacuoles in
the neutrophils of patients with an
LR attributed to an
infection.

CML

In CML, there are

more immature
cells, absolute
basophilia , and
Eosinophils.

CNL
Marked
neutrophilia with
fewer
metamyelocytes
and myelocytes
(<5%).
Immature
granulocytes:
promyelocytes5,
myelocytes,
metamyelocytes are
10 %
Blasts 1% .

Leukemoid
reaction

Peripheral Mature
blood neutrophils,
marked "left shift"

Bone Myeloid
marrow hyperplasia,
orderly
maturation, normal
morphology

LAP sCore High

CML CNL

Immature cells, Marked

basophils, and neutrophilia, no
eosinophils immature cells

Basophilia, Similar
eosinophilia, morphology witth
monocytosis, slight LR, packed bone
increase in blasts marrow, slight
and reticulin increase in
fibrosis. reticulin

Low High