Professional Documents
Culture Documents
Lecture 17_Lymphoma 53
Lecture 18_Bleeding Disorders 117
Lecture 15_Introduction to Hematology _ Iron Def Anemia 173
DLA_Video_Lecture_B12 _ Folic Acid Deficiency Anemia 226
DLA_Video_Lecture_Thalassemia _ Sideroblastic anemia 256
DLA_Notes on Normocytic Anemias 288
DLA_Notes on Thrombocytopenia 371
DLA_Notes_Characteristics of common Lukemias_Robbins Table 417
Pathophysiology Lecture 16
Topic: Introduction to white cell
disorders & Leukemias
DR. R. Tey
1
Lecture Outline
• Understand the role of genetic abnormalities in the
pathogenesis of leukemia
• Classification of white cell proliferation disorders
– Relationship between leukemia and lymphoma
– Relationship between aggressiveness of disease and prognosis
• Typical cases of individual types of leukemia: Clinical and
pathological features, treatment and prognosis
– Acute myeloid leukemia
– Acute lymphoblastic leukemia
– Chronic lymphocytic leukemia
– Chronic myeloid leukemia
• Describe different types of lymphoma, understand their
distinguishing pathological, clinical & diagnostic findings.
• Distinguish Myeloproliferative disorders, polycythemia vera &
Mylodysplastic syndromes and describe their presentations.
2
White Blood Cell Disorders
• Functional Abnormalities
– Rare
– Congenital
• Abnormal Proliferation
– Leukemia and lymphoma : “Cancers” of blood forming
cells, lymphocytes and related cells
– Other “clonal” disorders of blood cells:
• Myelodysplastic syndromes
• Chronic myeloproliferative neoplasms
3
Leukemia and Lymphoma :
Key Concepts
• There are many different types of leukemia and
lymphoma
• These are (usually) distinct diseases and not
morphological variations of the same disease
• The clinical course and treatment options in each
disease are often unique. Therefore correct diagnosis
is absolutely essential for correct management.
4
Leukemia and Lymphoma :
Key Concepts-2
• Some leukemias and lymphomas are “aggressive”
– They proliferate rapidly and, if untreated, are fatal within
weeks.
– Rapidly proliferating neoplasms are more susceptible to
chemotherapy and radiation. Some can be cured.
• Some leukemias and lymphomas are “indolent”
– They are slow growing and patients may survive months
to many years without treatment
– They do respond to modern treatments but tend to recur
or transform into aggressive forms.
5
Leukemia versus Lymphoma:
Operational Definitions
• When the abnormal lymphoid or myeloid cells are
present primarily in blood and bone marrow, it is
called leukemia
• When they are present as tumor masses (usually
lymphoid cells) in lymph nodes, spleen etc, they are
called lymphoma
• Some of these diseases may first present as leukemia
and later involve lymph nodes; others may present as
lymphoma and in the terminal stage have a leukemic
picture; some have both from the beginning.
6
KEY CONCEPT
• Many leukemias and lymphomas develop
as a result of specific chromosomal
translocations and/or gene mutations
• Because of these abnormalities the
affected cells show one or more effects:
– Rapid proliferation and/ or
– Lack of maturation and/ or
– Absence of apoptosis
7
How Genetic Abnormalities cause
Leukemia and Lymphoma
• Increased, uncontrolled, rapid proliferation
– Translocations involve oncogene
• Scenario 1 -
– The oncogene is over-expressed because it is moved next
to the promotor of another gene
– Example : Burkitt lymphoma
Oncogene – cMYC (on chrom. 8)
Promoter – immunoglobulin gene
(on chrom. 14)
8
9
How Genetic Abnormalities cause
Leukemia and Lymphoma
• Increased, uncontrolled, rapid proliferation
– Translocations involve oncogenes
• Scenario 2 -
– The oncogene becomes independent of other signals
(constitutional activation)
– Example : Chronic myeloid leukemia
Oncogene – ABL on chrom. 9
translocated to BCR region on
chrom. 22
10
11
How Genetic Abnormalities cause
Leukemia and Lymphoma
• Lack of maturation
– The affected genes are transcription factors involved in
normal maturation of myeloid cells
– The structure of the transcription factor is altered by
mutation or translocation
– The altered transcription factor cannot function
• Examples
– Translocation between chromosomes 15 and 17 involving
the transcription factor Retinoic Acid Receptor Alpha
(RARA)→acute promyelocytic leukemia gene (PML)
– Mutation: mutation of FLT3.
12
How Genetic Abnormalities cause
Leukemia and Lymphoma
• Prevent Apoptosis
– Cells do not die and accumulate to produce
tumors (lymphoma)
– Example: follicular lymphoma
14
Progress in defining the molecular landscape of AML
18
Cell of Origin
• Based on morphology and antigen expression
(“immunophenotype”) of abnormal cells, origin
from a normal cell in the hematopoietic or
immune (lymphoid) system is proposed
• Many biological properties (growth rate, response
to radiation, etc) of the leukemia or lymphoma
will be determined by the cell of origin and the
genetic abnormality present
– Example: BCR/ABL can cause either chronic myeloid
leukemia or acute lymphoblastic leukemia
19
Leukemias
• Main Possibilities:
– Acute vs Chronic (operational defn: weeks-months
vs months-years)
and
– Myeloid vs Lymphoid
• AML (80% adults, 20% children), CML (middle age adults)
ALL (80% children, 20% adults), CLL (older adults)
20
Case 1
• 41 Year old W/M
– In excellent health until 10 days ago
– Worsening breathlessness
– Noticed bleeding while brushing teeth
– No fevers, weight loss
– He previously worked in a warehouse for a carpet and cleaning
company. He was exposed to multiple soaps and furniture cleaning
agents.
• Lab
• Hemoglobin, 5.4 (low)
• Platelets, 12 (low)
• white count, 0.8 (low)
• Blood smear: severe pancytopenia
• Bone marrow performed
21
Normal promyelocyte
Bone marrow
22
FISH
23
24
Case 1
• FISH showed PML/RARA [t(15;17)]
abnormality
• Diagnosis: acute promyelocytic leukemia
(APML)
• A type of acute myeloid leukemia
• Must not be treated with the regular
chemotherapy regimen for AML
– Granules in leukemic cells may precipitate
severe, fatal DIC
25
Acute Leukemia - Treatment
• Supportive
• Treat infections
• Transfusions – platelets/ RBC’s
• Metabolic – fluid and electrolytes
• Definitive
• ATRA (all-Trans-Retinoic-Acid) or Arsenic Trioxide in
APML (drives maturation), followed by
• Cytotoxic chemotherapy
26
Acute Leukemia - Treatment
• Induction chemotherapy (anthracyline/
cytarabine)
• Consolidation/intensification
• Bone marrow (stem cell) transplant:
allogeneic (in some cases)
• Maintenance (in ALL)
27
AML- Prognosis
• Complete remission - 60 to 70%
• Approximately 25% of those achieving CR
survive 3 years (15% of patients)
• High dose chemotherapy- may be higher, up
to 30% survival
• With BMT - 30 to 40% may survive longer
28
Acute Myeloid Leukemias
• Several subtypes (WHO) based on
– Certain recurring genetic abnormalities, if
present, e.g., t(8;21), t(15;17), inv16, Flt
mutation3+/-; otherwise:
• Level and type of maturation
• AML can have maturation towards erythroid cells,
megakaryocytes, monocytes, granulocytes, etc;
designated AML M0 thru M7 (as in previous FAB
classification)
– Except for APML, all treated similarly but
prognosis may vary among the subtypes
29
Case 2
• Patient: 2 year old white female.
• Presentation:
– Fever
– Persistent cough unresponsive to antibiotics for 2
weeks
– Petechiae and ecchymoses
• Bone marrow exam and flow cytometry were
performed
30
• Diagnosis: B-lymphoblastic leukemia
31
Lymphoblastic Leukemia
32
Signs & Symptoms of Acute Leukemia
• Leukemic cells infiltrate
– Organomegaly, dysfunction
– Lymphadenopathy
– Hepatosplenomegaly
– Skin/gums
– CNS - confusion/decreased level of consciousness,
“leukostasis”
– Bone (rapid marrow expansion) - Pain
33
Signs & Symptoms of Acute
Leukemia
• Marrow failure due to replacement of normal
precursors by leukemic blasts
– decreased RBC: anemia, fatigue, etc
– decreased WBC: infections
– decreased platelets: hemorrhage
• Hyperviscosity (rare)
– seen in acute leukemias with extremely high WBC (>100k)
– More common in AML
34
Signs & Symptoms of Acute Leukemia
Hypermetabolic symptoms
• Fever (must always rule out infection first)
• Weight loss
• Hyperuricemia (breakdown of leukemic cells)
• Disseminated intravascular coagulation (DIC)
[especially with acute promyelocytic leukemia]
35
Diagnosis of Acute Leukemia
– Hx/PE are similar in AML and ALL
– A few blasts in peripheral blood are NOT diagnostic of acute
leukemia and lack of blasts does not rule out the diagnosis
– Bone marrow
• Morphology: Auer rods are patho-
gnomonic of AML (not present
in most cases); AML may have cyto- granules
plasmic granules
• Immunophenotyping (flow cytometry)
• Cytogenetics
• Special stains …
36
AML M5a, asp, MPO, 100x (SM-05-24920).jpg
Myeloperoxidase
38
DX: Chronic lymphocytic leukemia
CLL
Smudge cell
39
Diagnosis: CLL
• Flow Cytometry:
– CD5+, CD23+, monoclonal (k+ or ʎ+) B-cells
40
Epidemiology
• Most common type of leukemia in the
Western world
• 30% of all leukemias
• Male/Female = 2:1
• Relationship to “monoclonal B lymphocytosis
of uncertain clinical significance”? Precursor
CLL? Familial cases
41
Clinical Features of CLL
• Most patients asymptomatic in early stage
disease
• Commonly diagnosed as incidental
lymphocytosis in elderly population
• Some patients present with lymphadenopathy
(small lymphocytic lymphoma)
42
Symptoms in CLL Due to:
• Marrow failure - late stage disease
• Lymph node enlargement - cosmetic,
occassionally painful, rarely obstruction
• Hypogammaglobulinemia - lack of normal B
cells, recurrent infections
• Autoimmune hemolytic anemia/
thrombocytopenia
43
Prognosis in CLL
• Survival varies from <5 years to >25 years
– Prognostic factors predicting survival
• Cytogenetics
• Immunoglobulin (Ig) gene mutation status
• ZAP-70 expression
• CD38 expression
– Bad: presence of ZAP-70, CD38, chromosome 17p
abnomality, unmutated IgH gene status
– Good: IgH mutations, chromosome 13q abnormality
44
Case 4
• 42 y/o dentist
– Turned down as a blood donor because of Hgb of 11.5
• PE: Splenomegaly, 4cm below left costal margin
• Further testing revealed:
– WBC: 47,000/ cu mm (high)
– WBC diff: Neutrophils 40%
– Bands: 20%
– Metamyelocytes:16%
– Myelocytes: 8%
– Promyelocytes:6%
– Blasts: 2% Immature myeloid cells
– Eos: 2%
– Basos: 4%
– Monos: 2% Basophilia is characteristic
– Platelets: 680,000/ cu mm (sl. high)
45
Case: peripheral Blood Smear: Leukocytosis with Left
Shift in Maturation
Band
Myelocyte
Blast
Neutrophil Metamyelocyte
46
Case 4(cont.)
• Diagnostic evaluation:
Cytogenetics- Philadelphia chromosome +
– t(9;22)(q34;q11.2)
– ABL and BCR genes fused due to translocation
– Detected by conventional cytogenetics, FISH or PCR
• Diagnosis: Chronic myelogenous leukemia (CML)
47
Chronic Myelogenous Leukemia
(CML)
48
Natural Course of CML
• Chronic phase 2 to 4 yrs – variable (range
1-10 yrs)
– Cells maintain capacity to differentiate
• Accelerated phase
– Sharp increase in WBC count, basophil count,
or blasts (<20%) or development of anemia or
thrombocytopenia
• Blast crisis (blast phase) > 20% blasts
– Transforms to AML (80%) or ALL (20%)
49
Treatment
• Chemotherapy (busulfan, hydroxyurea) can lower
WBC, alleviate symptoms but has no effect on
long term survival.
• Interferon may prolong chronic phase
• Tyrosine kinase inhibitors –imatinib and newer
(second and third generation) TKIs.
– “ targeted therapy”
– Majority of patients achieve cytogenetic and molecular
response; managed as chronic disease?
• Only curative therapy to date is allogeneic bone
marrow (stem cell) transplant.
– Possible to reach lasting remission with TKIs?
50
Imatinib (Gleevec)
51
Pathophysiology Lecture 17
Topic: Lymphomas & Chronic
Myeloproliferative disorders
DR. R. Tey
1
Learning Objectives
• Describe different types of lymphoma, their
distinguishing pathological, clinical & diagnostic
findings.
• Distinguish Myeloproliferative disorders and describe
their presentations.
2
Lymphomas
3
Lymphomas
Non-Hodgkin
Hodgkin Lymphoma
Lymphoma (NHL)
(HL)
4
B-and T-cells in Normal lymph
Nodes
Lymphoid
Follicle
Sinusoids
5
Morphological Patterns
• Overall Pattern
– Diffuse versus follicular vs “nodular”
– Monomorphic versus polymorphic
• Cell Morphology
– Small cells versus large cells
– Irregular (“cleaved”) versus round/oval nuclei
– Condensed, corse chromatin versus “blastic”
(immature)
• Other Features
– Fibrosis or necrosis
– Reactive cells (e.g., macrophages, granulocytes)
6
Morphological Types
• Overall Pattern
– Diffuse versus follicular versus nodular
7
Morphological Types
– Monomorphic versus polymorphic
“Blastic”(immature)
Small Cells
Cleaved = centrocytes
Large Cells
9
B-cell, SLL Prolif Center, 130x
Clonality in Lymphomas
• Like all cancers, lymphomas are clonal
processes, i.e., progeny of one abnormal
cell
• Monoclonality can be established in B-cells
by showing that all cells express one type
(either kappa or lambda) of Ig light chain
– Flow cytometry detects surface
expression
– Immunohistochemistry for plasma cells
(cytoplasmic)
10
Clonality in Lymphomas
• Definitive T-cell clonality is established by
showing clonal rearrangement of the T-cell
antigen receptor gene by molecular
methods (pcr based)
• In Hodgkin lymphoma, most cells in the
lesion are reactive; only a few clonal
abnormal cells are present. Clonality can be
established only after microdissection of
the abnormal cells, which are of B-cell
origin (not a routine diagnostic procedure)
11
Which of the following statements about
lymphoid malignancies is TRUE?
:7212
Common Immunophenotypes of B-
Cell Lymphomas
• CD5+
– Chronic lymphocytic leukemia/small
lymphocytic lymphoma (CLL / SLL)
– Mantle cell lymphoma
• CD10+
– Follicular lymphoma
– Burkitt lymphoma
– Lymphoblastic lymphoma (precursor B-cells;
form of ALL)
• Negative for CD5 and CD10: (MZ, LPL)
13
CD5+ B-cell Lymphomas
CLL / SLL Mantle Cell
Immunopheo- CD23+ CD23- (most)
type
Cyclin D1 Negative Positive
Morphology-1 Proliferation Monotonous
centers
Morphology-2 Diffuse or pseudo- Mantle zone, nodular,
follicular or diffuse patterns
Prognostic IgH mutation, Blastic variant
Markers CD38, ZAP-70
Cytogenetics Abnormalities of 11, t(11;14)(q13;q32)
12, 13, 17
14
B-cell, SLL diffuse, 3x
CLL / SLL
15
B-cell, SLL Prolif Center, 130x
Mantle Cell Lymphoma
(MCL)
Cyclin D1 MCL
17
B-cell, MCL blastoid, leukemic, Asp, 100x (SM-06-7019)
CD10+ B-cell Lymphomas: Follicular and
Burkitt
• Follicular lymphoma
– An indolent but currently incurable lymphoma
of older adults
– Second commonest NHL in the West
– Specific cytogenetic abnormality present:
t(14;18)(q32;q21)→overexpression of bcl-2
protein→antiapoptosis effect (cells don’t die)
18
Follicular Lymphoma
Follicular lymphoma
19
Microscopic: Follicular Lymphoma
20
Follicular Lymphoma: Immunohistochemistry
t(14;18)→Bcl-2 over-expression→lymphoma
persists
21
Bcl-2 positive: Follicular lymphoma
CD10+ B-cell Lymphomas: Follicular and
Burkitt
• Burkitt lymphoma
– Extremely aggressive lymphoma associated
with rearrangement of the c-myc oncogene
[t(8;14)(q24;q32)]→ uncontrolled, rapid cell
growth
– Endemic, sporadic, and immunodeficiency-
associated variants
22
Dennis
Burkitt
23
Burkitt Lymphoma
24
Burkitt Lymphoma
Microscopic
Lymph node
“Starry sky”
Bone marrow
25
More B-Cell Lymphomas/Neoplasms
• CD5-, CD10-
– Small to medium size cells
Marginal zone lymphomas
– Extra-nodal, MALT type (most common type)
Hairy cell leukemia
Lymphoplasmacytic lymphoma (most are associated with
Waldenstrom’s macroglobulinemia)
– Large Cell
Most Diffuse Large B-Cell Lymphomas (DLBCL)
• CD138+ , CD20-
– Plasma cell neoplasms
26
Diffuse Large B-Cell Lymphoma
(DLBCL)
27
DLBCL
• Heterogeneous group
– Morphologic variation
e.g.T-cell/histiocyte rich (and others)
28
DLBCL: Morphology
29
DLBCL
• Prognostic Groups
– Based on gene expression array analysis
31
MALT = Mucosa Associated
Lymphoid Tissue
• Chronic inflammation, such as chronic gastritis
due to H pylori, leads to development of reactive
lymphoid tissue at these sites
• In some patients, a low grade lymphoma develops
= called extra nodal marginal zone lymphoma of
mucosal associated lymphoid tissue or MALT
lymphoma
• Initially the lymphoma may be curable by
treatment of the infection (removal of the
stimulus)
32
Extra-Nodal Lymphomas and MALT
Lymphomas
33
Plasma Cell Neoplasms
• Plasma cells are terminally differentiated B cells specializing in
immunoglobulin (antibody) production
• Plasma cell myeloma (“multiple myeloma”)
– Disease of older adults
– involves multiple bone marrow sites, producing lytic bone lesions
– Releases monoclonal immunoglobulin (often called “paraprotein” or
“M-spike) into blood and / or urine
– Paraprotein may secondarily affect kidneys, cause amyloid deposition,
etc
– Indolent but incurable disease
34
Multiple
myeloma
35
Properties of T-Cells
• Mature in thymus from precursor T-cells
that derive from bone marrow; this process
eliminates “self-reactive” T-cells
36
Properties of T-Cells
• T-cell antigen receptor (TCR) on the cell
surface
– Either αβ (95%) or γδ (5%); both types associated
with a signal transduction molecule, CD3
37
T-cell Lymphomas
• Uncommon in the West : about 10% of NHL
• Many are site specific: Examples
• Skin: mycosis fungoides (most common), primary
cutaneous anaplastic large cell, subcutaneous
panniculitis-like, etc.
• GI Tract: enteropathy type,
hepatosplenic T-cell
38
T-cell Lymphomas
39
Example: Mycosis Fungoides- A
Cutaneous T-Cell Lymphoma
• Patches and plaques in skin for many years
– Epidermotropic mature T-cells with “cerebriform” nuclei
– Typically CD3+, CD4+, CD2+, CD5+, but CD7-
• Sometimes associated with Sezary syndrome:
generalized aggressive disease with erythroderma
and circulating neoplastic cells
40
A,B. Mycosis fungoides, patch stage
“Cerebriform” cells
41
Pautrier’s microabscesses
42
Mycosis fungoides, 35x, (Duke)
Hodgkin Lymphoma
• About 1 in 7 lymphomas are Hodgkin
lymphomas in the US
• Occurs in young adults with a second peak in
older adults
• Highly curable, even in late stage disease, with
chemo/ radiation therapy (only 1 in 20
lymphoma deaths).
43
Hodgkin Lymphoma
• Two types:
– Classical HL (95%)
– Nodular Lymphocyte-predominant HL (5%)
– The abnormal cells are called “Reed-Sternberg”
cells and “popcorn” or “L&H” cells , respectively
44
Classical HL – Nodular Sclerosis type
HD, NSHD, 3X
45
Classification-WHO 2008 Lymphomas
Mixed
Aggressive / Indolent /
Cellularity Extra-Nodal Nodal
High Grade Low Grade
Lymphocyte
Myecosis Fungoides Anaplastic Large Cell
Marginal Zone
Rich
Diffuse Large
Subcut Panniculitis Like Lymphoplasmacytic
B-Cell Lymphoma
47
Chronic Myeloproliferative
Neoplasms (MPN)
• Chronic myelogenous leukemia (CML) – ↑Myeloid
cells (mature > immature)
• Polycythemia vera (PV) - ↑RBC (also WBC and
platelets but to lesser extent than RBC)
• Essential thrombocythemia (ET) - ↑Plt
• Primary myelofibrosis (MF) - ↑Fibrosis
48
Chronic Myelogenous Leukemia
(see Previous Lecture)
• Clonal disorder of hematopoiesis that arises from a
mutation in stem cell or early progenitor
• Dysregulated production of mostly mature
differentiated cells of myeloid lineage
• Propensity to transform into acute leukemia
• Acquired genetic mutation
• t(9;22) [BCR-ABL]→chronic myeloid leukemia
49
Chronic Myeloproliferative
Neoplasms: Clinical Features
• Enlarged spleen (except in essential thrombocythemia)
• Present with abnormal WBC, RBC, or platelet count
• Thrombosis and bleeding → ? platelet dysfunction
• Must be distinguished from a reactive state, i.e.,
– RBC → due to hypoxic stimulation with excess
erythropoietin
– Plts → due to infection, inflammation
– WBC → due to infection, inflammation
• Natural history evolves over years, i.e., not acute
• Usually NOT associated with fever, night sweats, etc
50
51
Molecular Abnormality in MPN
• JAK2 Mutation:
– Janus-like Kinase-2
– A tyrosine kinase
– Mutated in over 70% of polycythemia vera and lower proportions
of other MPN; if +, strong evidence for a MPN
• Mutations of MPL and Calreticulin Receptor noted in
varying proportion of MPNs
• In the absence of disease defining mutations, rule out
reactive conditions first
• Always rule out CML, because it has a specific treatment
(TK inhibitors,e.g. imatinib).
52
Case
• 50-year-old male smoker
• Chief complaint
– fatigue, dizziness
– itching after bath
• On examination
– plethoric “ruddy” complexion
– splenomegaly, moderate
• CBC: Hgb – 22.0 (high), WBC – 14 (high), Plt – 550 (high
nl)
• Questions
– 1. Is this a reactive erythrocytosis or PV?
– 2. If this is PV, what are treatment options?
– 3. What are complications of PV?
53
PV vs Reactive Polycythemia vs Reduced
Plasma Volume
• Diagnostic Criteria
– RBC mass - 36 ml/kg for males
- 32 ml/kg for female
• R/O relative polycythemia (altitude, smoking, COPD,
etc)
– Splenomegaly (congestion, functional
hypertrophy; usually not as large as in MPN)
– R/O CML (definitive test = bcr-abl translocation)
– Serum B12 (due to transcobalamine III
caused by WBC)
– JAK-2 mutation – Definitive diagnosis
54
JAK-2 mutation results in activation of JAK-STAT
pathway in absence of ligand – “cytokine
independent constitutive activation”
55
JAK-2 protein- important for signal
transduction of multiple cytokines
56
Treatment of PV
• Improve Symptoms
– Hyperviscosity → need to Hgb (RBC) → phlebotomy
• Caution: Monitor for iron deficiency
• Other Symptoms
– Weight loss/sweating
– Splenomegaly
• chemotherapy ?
• splenectomy
– Thromboembolism (aspirin, other?)
– Bleeding
• Therapy targeted at JAK-2 (JAK-2 inhibitors currently in
clinical trials, with some promising results)
57
PV Case: Progression
• Synopsis
– Diagnosed in 1982 → PV
– Treated with phlebotomy and P32
• Returns 1990
– fatigue/splenomegaly/weight loss
– CBC Hgb 5.2 (low)
Teardrop cells
Plts 50k (low)
Nucleated RBC
Immature myeloid cells
WBC 2.0 (low)
58
Peripheral blood smear
59
Teardrop cells = splenic
hematopoiesis
60
Reticulin fibrosis
61
Myelofibrosis: Primary vs Secondary
• Myelofibrosis (MF) may arise from a
preexisting MPN, like polycythemia vera in the
preceeding example
• Except by history, secondary MF and primary
MF can not be differentiated
62
Myelofibrosis
• Cardinal Features
– Marrow fibrosis
• Cytokine production by abnormal myeloid cells stimulates
normal fibroblasts→ reticulin fibrosis
– Massive splenomegaly due to myeloid metaplasia
(hematopoiesis in the spleen)
– Leukoerythroblastic blood picture
• Nucleated RBC
• Immature WBC
• Natural Course
– Progressive marrow failure (70%) or AML (30%)
– Survival 2-5 years
63
Essential Thrombocythemia (ET)
64
Pathophysiology lecture 18
Topic: Bleeding Disorders
Dr Tey
Hemostasis
• Primary
• VWF, platelets, fibrinogen adhere to injured vessel wall →platelet plug (stops
the “leak”)
• Secondary
• Coagulation cascade
• Thrombin generation at site of injury (platelet plug for localized activation)
• Generation of fibrin clot
• Tertiary
• Crosslinking of fibrin strands
• Clot maturation, dissolution, wound healing
2
Primary Hemostasis at the site of injury
• Platelet adhesion to vessel wall
• GP1b (1b-V-IX complex),VWF and collagen in vessel wall
• Platelet activation
• Shape change
• Release of alpha and dense granule contents
• Thromboxane A2 generation (facilitates platelet aggregation)
• Platelet aggregation
• Platelet/platelet interactions require fibrinogen and GPIIb/IIIa receptor on
platelets
3
Secondary Hemostasis
• Activation of clotting cascade
• Requires activated platelets+phosopholipid surface
• Tissue factor (protein in subendothelium)/VIIa key to initial activation of
clotting cascade
• Inactive zymogens (clotting factors) → activated clotting factors
• Generate fibrin clot
4
Tertiary Hemostasis
• Stabilization of fibrin clot
• Thrombin generates fibrin monomers, a “loose clot”
• Fibrin monomers crosslinked by factor XIII→stable fibrin clot
• Fibrinolysis
• tPA (tissue plasminogen activator) converts plasminogen to plasmin which
degrades clot
• PAI -1 (plasminogen activator inhibitor) – inhibits plasminogen activation
• Alpha 2 antiplasmin – inhibits plasmin
5
Understanding the Coagulation Cascade
• Basic requirement:
• Convert fibrinogen to fibrin rapidly at the site of vascular
injury but prevent this from happening everywhere else
• Special challenge:
• Performing the biochemical reactions at a particular site
while the surrounding blood is constantly moving
• Solutions:
• Stepwise reaction which is triggered by product of injury
and then amplifies rapidly
• Tight control by inhibitors
• Anchor the reaction on a solid surface
6
Understanding the Coagulation Cascade
• The cascade was proposed based on results of two
coagulation tests in patients with bleeding disorders
• Prothrombin Time (PT)
• Plasma clots in a few seconds (10 to 12 sec) when mixed
with calcium and tissue extract (phospholidid source)
• The test is prolonged in patients with some bleeding
disorders but is normal in Hemophilia
• NOTE: To maintain uniformity of test results between
different laboratories, a standardized ratio of PT result in
the patient and a normal control is often used. This is
called INR (International Normalized Ratio).
7
Understanding the Coagulation Cascade
• Activated Partial Thromboplastin Time (aPTT)
• Plasma, preactivated with clay (kaolin), clots in about 40 sec when mixed with
calcium and phospholipids
• The test is prolonged in Hemophilia
• Two independent starting points proposed
1. Activated by tissue factor – “extrinsic” to the vessel
2. Activated on endothelium – “intrinsic” to the vessel
8
The Traditional Coagulation Cascade
Tissue Factor
Factor VIIIa
Fibrinogen Fibrin
9
The Traditional Coagulation Cascade
Fibrinogen Fibrin
10
Coagulation Cascade
• Coagulation factors are serine proteases or their cofactors
• Vitamin K dependent factors (II, VII, IX, X) and factors XI and XII are
the proteases
• NOTE: The antigoagulant factor, Protein C, is also vitamin K dependent
• V, VIII, tissue factor, and low molecular weight kininogen are cofactors
• Synthesis by liver – all except VIII (endothelium)
• Calcium is cofactor for many reactions
11
Coagulation Cascade:
The central conundrum
12
The Connection
• A small amount of thrombin is generated from the “extrinsic
pathway”
• Thrombin activates the cofactors factor VIII and factor V
• The cofactors increase the rate of the reaction and allow enough
thrombin to be generated even in a moving blood stream
13
The Traditional Coagulation Cascade
Prothrombin Thrombin
Fibrinogen Fibrin
14
Relative Rate
Prothrombinase of Prothrombin
Components Activation
-Thrombin
Prothrombin
FXa
1
Ca2+FXa
FVa HC
Ca2+
Ca2+ FXa
Ca2+
30
FVa LC
Ca2+ FXa
FVa HC
FVa LC
300
Prothrombinase
Ca2+ FXa
FVa HC 2+
Ca
FVa LC
300,000
15
The true value of PT and aPTT
• These tests may NOT predict which patient will bleed during surgery
• They are valuable in monitoring patients treated chronically with
anticoagulant
• Heparin treatment is monitored with aPTT
• Coumadin treatment is monitored with PT
16
Mode of Action of Anticoagulants
17
Bleeding disorders: Congenital
• The commonest deficiencies are in Factor VIII and IX (hemophilia A
and B, respectively) and von Willebrand Factor
• Factor V and VII deficiencies are rare but cause severe bleeding.
18
Incidence of Congenital Bleeding Disorders
• Hemophilia A (F VIII) 1 in 5000 – 10,000
• Hemophilia B (F IX) 1 in 30,000
• vW Disease: Mild 1 in 100
Severe 1 in 10,000
• Factor VII def 1 in 500,000
• Factor V def 1 in 1 million
19
Inheritance
• Hemophilia A and B
• X linked recessive
• 30% spontaneous (new) mutations
• von Willebrand Disease
• Type 1 and 2 are autosomal dominant
• Type 3 (severe) is autosomal recessive
• FV and FVII deficiencies
• autosomal recessive
20
Acquired Hemophilia
• Autoimmune disorder with antibodies (inhibitors) to specific
coagulation factors
• Factor VIII inhibitors most common
• Precipitating conditions
• Pregnancy
• Post-partum
• Other autoimmune disorders (lupus, rheumatoid arthritis, myasthenia gravis)
• Those with congenital absence of the factors may develop inhibitors
when treated with the factor
21
Acquired Hemophilia
Clinical Presentation
• Usually dramatic and sudden severe bleeding
• A survey found 87% of these patients had a major bleed with 22%
mortality
• Treatment
• Recombinant F VIIa (enhances extrinsic pathway so as to bypass the need for
F VIII of the intrinsic pathway)
• Early recognition and treatment is key
22
The Traditional Coagulation Cascade
Fibrinogen Fibrin
23
Case presentation
24
Laboratory Data
• ABC normal, including platelet count of 233,000/ml
• Serum chemistries all normal.
• PT 11.9 sec. aPTT 72.6 sec. (~2X normal)
• Repeated coagulation studies: unchanged.
25
Key Concept
• A coagulation factor present at 50% or greater of normal level does
not appreciably prolong the coagulation test (PT or aPTT, as
appropriate).
27
Incubated Mix
• Mix patient plasma with normal pooled plasma
(50:50).
• The factor which is completely absent in patient plasma
will be replenished by the normal plasma to a 50%
level.
• All other factors will be at 100% level.
• If due to factor deficiency, prolonged aPTT will
correct by mixing.
• If an inhibitor is present, prolonged aPTT will not
correct.
28
Evaluation for a factor deficiency
• If the incubated mix completely corrects, what do
you check next?
• Factor VIII deficiency: bleeds.
• Factor IX deficiency: bleeds.
• Factor XI deficiency: bleeds.
29
Abnormal mixing study
30
Abnormal mixing study
31
Lupus “Anticoagulant”
• Lupus anticoagulant (also known as lupus antibody, LA, or lupus
inhibitors) is an immunoglobulin that binds to phospholipids and
proteins associated with the cell membrane. The name is a misnomer.
LA is actually a prothrombotic agent; the presence of LA antibodies
precipitates the formation of thrombi in vivo.
32
Lupus “Anticoagulant”
• The presence of these antibodies causes an increase in the PTT. It is
speculated that the presence of the antibodies interferes with
phospholipids utilized to induce in vitro coagulation.
• In vivo, it is thought to interact with platelet membrane
phospholipids, increasing adhesion and aggregation of platelets; thus
it is prothrombotic in its actions in vivo.
33
Types of Bleeding
34
Types of Bleeding- Hemophilia
• Joints: hemarthrosis
• Hemophilic arthropathy
• Muscles: hematomas of calf, iliopsoas, forearm
• Complications: compartment syndrome with neurovascular compromise
• CNS bleeding: number one cause of death
• Treat first then investigate
35
Hemarthrosis
36
Hemarthrosis
37
Von Willebrand Disease
• Autosomal inheritance
• Most common inherited bleeding disorder
• 1% of general population
• vWF: large complex factors assembled into mulitimers
• Found in platelets (alpha granules) and endothelial cells
• Carrier molecule for factor VIII
• Binds platelets to endothelium under high shear stress via Gp1b
• Mucocutaneous bleeding: epistaxsis, gums, menorrhagia
38
Von Willebrand’s Disease
39
Platelet Disorders
• Quantitative or qualitative defects
• Congenital or acquired
• Congenital – uncommon
• defects in adhesion, aggregation or loss of specific granule
• Acquired – commonly drug related, eg., aspirin
• Patients seldom bleed spontaneously when platelet
count >50k unless they have a functional defect in
platelets
40
Diagnosis
• Take a history!
• Type of bleeding
• Drugs
• Family history
• Laboratory tests
• “Screening” [INR (PT), aPTT, platelet count]
• Specific factor assays (VIII, IX, vWF)
• Platelet function studies
• “Bleeding time” – non specific, insensitive, poorly
reproducible, non predictive of bleeding; don’t order this!
41
Increased aPTT
• No bleeding
• Heparin contamination or therapy
• Lupus anticoagulant ( antibody against phospholipid reagent in vitro)
• Factor XII deficiency
• Bleeding
• Hemophilia A ( VIII) or B (IX)
• Less common Factor XI
42
Increased INR (i.e., prolonged PT)
• Vitamin K deficiency
• Coumadin (Warfarin) therapy
• Liver disease
• Rarely, factor VII deficiency
43
Increased INR and aPTT
• Liver disease (multiple factor deficiency)
• Excessive warfarin therapy or severe Vit K deficiency [II
(prothrombin), VII, IX, X]
• Warfarin and unfractionated heparin
• DIC (also have low fibrinogen)
• Isolated factor deficiency in common pathway (X, V, II, fibrinogen)
44
Bleeding with Normal INR/PTT
• vWD – check vW Factor levels
• Platelet function defect
• Factor XIII deficiency (required to crosslink fibrin to stabilize clot)
classically bleeding from umbilical stump after delivery.
45
Hyper-Coagulable States or
“Thrombophilia”
46
Hyper-Coagulable States or
“Thrombophilia”
47
Hyper-Coagulable States
• Watch for hypercoagulability/ thrombophilia in post-operative
patients, cancer patients, inflammatory conditions, pregnancy, and in
patients confined to bed.
• A battery of tests is required to diagnose the presence and nature of
hyercoagulable states
• A specific abnormality may not be seen.
• Patients with a demonstrated abnormality or episode of thrombosis
may need long term anticoagulation to avoid further episodes of
thrombosis.
48
Normal Control of Coagulation
• Protein C and its cofactor Protein S
• Physiologically most important anticoagulant system
• Point mutation in F V (called F V Leiden) is prevalent in northern European
countries; prevents protein C from binding and inhibiting coagulation)
• Thrombosis risk 2-3x in heterozygous and 8-10x higher in homozygous state
• NOTE: Protein C and S are vitamin K dependent factors
49
Normal Control of Coagulation
• Antithrombin
• A serine protease inhibitor (ser p in) which inhibits thrombin and also factors
VII, IX, X
• Heparin acts by potentiating the action of antithrombin 1000 fold
• Tissue factor pathway inhibitor
50
Classification of Inherited Disorders of Platelet Function
51
52
Inherited Defect of Platelet Adhesion
• Bernard-Soulier Syndrome
• Rare, autosomal recessive
• Abnormality in GPIb/V/IX complex
• Bleeding time is markedly prolonged, platelet counts
moderately decreased, platelets are large on peripheral
smear
• Aggregation studies
• Response to ristocetin is decreased or absent
• Similar to vWD, but not corrected by normal plasma
• Response to ADP, epinephrine, thrombin, and collagen is
normal
53
Platelet
Aggregometry
• Glanzmann Thrombasthenia
• Rare, autosomal recessive
• more severe mucocutaneous bleeding manifestations than most platelet function
disorders
• Quantitative or qualitative defect in the GPIIb/IIIa complex
• Platelets are normal in number and appearance, but remain separate on
blood film
• Aggregation studies – very low to absent response to all agonists, except
ristocetin
55
Acquired defects of platelet function
• Vastly more common
• Causes
• Intrinsic to platelets
• Myeloid neoplasms – MDS, ET, etc
• Extrinsic
• Uremia
• Dysproteinemia
• Anti-platelet antibodies
• Drugs
• Aspirin
• NSAIDs
• And many others
56
Pathophysiology Lecture 15
Topics: Introduction to Hematology
&
Microcytic Anemia
Dr. R. Tey
You are required to study ALL DLAs
on Hematology posted in sakai
General Objectives
• Describe the normal constituents of blood
• Gain a general understanding of hematopoiesis and definition of a
stem cell
• Understand the molecular mechanism of a hematologic disease and
the implications for treatment
• Describe the diseases affecting each constituent of blood
• Develop a diagnostic approach to common hematologic problems
3
Total Blood in Adult ~ 5 liters
Cells = 45%
4
Constituents of Blood
• Cellular component (45%)
• Red blood cells (erythrocytes)
• White blood cells (leukocytes)
• Platelets (thrombocytes)
• Plasma or fluid component (55%)
• Water (95%)
• Proteins (albumin, gamma globulins, clotting factors)
• Electrolytes (Na, Cl, K, etc. )
• Glucose, fats and minerals
• Serum = The liquid portion after blood clots
5
Diseases of Blood: What can go wrong
with each component?
Too little – reduced, absent or dysfunctional
• RBC → anemia → fatigue
• WBC → leukopenia (neutropenia, lymphopenia etc.) → infection
• Platelets → thrombocytopenia → bleeding
• Clotting factors → hemophilia → bleeding
6
Diseases of Blood - What can go wrong with each
component?
• Too much: Cellular components – normal physiologic
response, abnormal clonal (malignant) proliferation or
abnormal function
• RBC → erythrocytosis (response to hypoxia or polycythemia rubra
vera)
• WBC → leukocytosis (response to infection or leukemia)
• Platelets → thrombocytosis (response to inflammation or essential
thrombocytosis)
• Clotting factors → (abnormal function) hypercoagulable states
7
Peripheral Blood Cells :
Some Basic Facts
Life
Number / Produced Destroyed
Cell Type Span in
cmm in in
Days
Red Cells* 5 x 106 120 BM Spleen
8
Diseases of the Blood - Overview
• Red Blood Cells
• Anemias
• Microcytic, macrocytic and normocytic: based on RBC size
• White Blood Cells
• Leukemias (acute and chronic)
• Chronic myeloproliferative neoplasms
• Myelodysplastic syndromes
• Chronic lymphoproliferative diseases (lymphomas)
• Platelets/Clotting factors
• Thrombocytopenia and thrombocytosis
• Hemophilia and other congenital bleeding disorders
• Thrombosis and hypercoagulable states
9
How are Hematologic Disorders Diagnosed?
• History
• Screening tests
• Special / confirmatory tests
10
Screening Lab Tests for Hematological Disorders
11
Peripheral Blood Smear
Platelet
Eosinophil
Band
Neutrophil
Lymphocyte
Basophil
Monocyte
12
Blood – Where Is It Produced?
• In utero
• Yolk sac is the first site of hematopoesis
• Liver/spleen after 5-6 weeks gestation and remains a major
site of production until third trimester
• Bone marrow development starts at 3 months and
becomes the major site by 5-6 months gestation
13
Stem Cells
• Adult Stem Cells
• Definition:
• Undifferentiated cells, found throughout the body after embryonic
development, that multiply by cell division to replenish dying cells
and regenerate damaged tissues
• Properties:
• Self-renew indefinitely
• Generate all the cell types of the organ from which they originate =
differentiation
• Potentially regenerate the entire organ from a few cells.
14
Hematopoietic Stem Cells
• Production maintained by a small pool of stem cells ( normally
<0.1% of marrow, 0.01% in peripheral blood ).
• Pluripotent (capacity to generate all cell lineages).
• Capacity for self renewal ( “immortal”).
15
Hematopoietic Stem Cell
• stem cell (pluripotent, quiescent) → colony forming unit (CFU; high proliferative rate,
multilineage potential, limited self renewal) →committed CFU (high proliferative rate, lineage
specific)
16
Production of Blood Cells
17
Regulation of Hematopoiesis
• Cell turnover in adult is normally approximately 1 trillion cells/24 hrs
• Under conditions of “stress”, marrow responds to cytokines (growth
factors) by increasing one or more cell lineages
• Hypoxia → erythropoietin → RBC
• Infection → G-CSF and/or GM-CSF → neutrophils/monocytes
• Hemorrhage → thrombopoietin → platelets
18
How are hematopoietic cells identified?
• Morphology
19
How are hematopoietic cells identified?
• Immunophenotype
• Each cell type has a unique combination of antigens expressed on the surface
• The cell surface antigens are designated as CD followed by a number, eg, CD1,
CD2, CD45 (common leukocyte antigen), CD71 (transferrin receptor) etc.
• Flow cytometry is used on fresh specimens (bone marrow, blood, lymph nodes,
etc.) to define the set of antigens expressed on a particular cell type
• Immunohistochemistry can be used on fixed tissues to define antigen expression
20
Antigens on Normal Populations
• Myeloid:
• Blasts (precursor cells): CD34, CD117, HLA-DR, low CD45
• Grans: CD13>CD33, myeloperoxidase (MPO)
• Monocytes: CD33>CD13, CD4, CD14
• Erythroid
• CD71, CD235 (glycophorin A)
• Lymphoid
• B-cells: CD19, CD20, CD22, CD79a, sIg (κ or λ), CD23, CD10
• Plasma cells: CD19, CD38, CD138, (CD45-, sIg-, cIg+)
• T-cells:
• Mature: sCD3, CD2, CD4 or CD8, CD5, CD7, CD56, TCR-αβ (>95%) or γδ (less than 5% in blood)
• Immature: CD1a, TdT, CD4+CD8
• NK-cells: sCD3 neg, CD16, CD56, CD57, CD158a, b or c
21
Stem Cell – Clinical Correlates
• Stem cell loss or failure
• Aplastic anemia
• Loss or reduction of a specific committed CFU
• Pure red cell aplasia
• Chronic neutropenia
• Stem cell transplantation
22
Anemia : Definition
• Anemia is, strictly speaking, reduction in red cell mass
• But for all practical purposes anemia is defined as a level of
hemoglobin/ hematocrit below normal for age and sex
23
Classification of Anemias
Morphologic classification
• Normocytic vs Microcytic vs Macrocytic (Size based)
• Normochromic vs Hypochromic (Hb content based)
Etiologic Classification :
Decreased production of red cells
• Not enough raw material
• Dietary deficiency
• Transport problem
• Congenital abnormalities of synthesis
• Decreased/ defective progenitor cells
Defects of red cell survival
• Red cell membrane abnormalities
• Hemoglobin abnormalities
• Red cell enzyme abnormalites
• Vascular and other extrinsic causes
Sequestration of red cells
• Splenic enlargement/hypersplenism
24
Red Cell Indices
Normal Decreased below Increased above
Range lower limit = upper limit =
Hgb, g/dL M 14 - 18
Anemia Polycythemia
F 12 - 16
26
CASE 1 – Microcytosis
• 27 year old woman from South Africa
• Referred to Hematology
Anemia
Microcytic
28
Case 1 - contd
29
Peripheral blood smear:
Microcytic, hypochromic anemia
Hypochromic
Anisocytosis
(RDW= 18.6)
Poikilocytosis
30
Microcytic Anemias
• Definition
• MCV <79 fL in an untransfused person (transfused cells can
give a false normal value for the MCV)
• Causes
• Decreased globin synthesis (thalassemias and
hemoglobinopathies)
• Decreased heme synthesis
• iron deficiency
• anemia of chronic inflammation/disease
• sideroblastic anemia
31
Microcytic Anemias
• Mechanism
• Synthesis of hemoglobin is decreased
• Cell division of precursors proceeds at a faster pace than
Hgb synthesis.
• Result is pale (hypochromic), small (microcytic)
erythrocytes.
• If the defect is severe, erythrocytes may be destroyed in
the bone marrow (intramedullary hemolysis).
32
Microcytic Anemias
Approach
• Assessment of iron stores
• bone marrow aspiration/biopsy (direct method)
• serum ferritin (indirect method)
• serum iron, Total Iron Binding Capacity (TIBC) and transferrin
• examination of blood film to confirm microcytosis and other
morphological changes of iron deficiency
33
Iron Deficiency
History
• Iron intake (diet)
• Iron loss (bleeding, pregnancy, hemolysis)
• Effects of decreased O2 delivery (fatigue, cardiorespiratory compromise)
• Effects of iron deficiency (pica, other end-organ effects)
34
Iron Deficiency
• Physical Examination
• effects of anemia: pallor, CHF, tachycardia, heart murmurs
• effects of iron deficiency: hair, nails, tongue
• Investigations
• confirm anemia, iron deficiency
• search for occult blood loss: rectal exam, stool testing for blood
35
Clinical Manifestations of IDA
• Glossitis: smooth painful tongue secondary to papillary atrophy
• Cheilitis: angular stomatitis
• Koilonychia: thinning, flattening and spooning of the nail
• Pica:
• Pagophagia (compulsive ice eating)
• Amylophagia (compulsive starch eating)
• Geophagia (compulsive dirt eating)
36
Clinical Manifestations of IDA
Glossitis Koilonychia
37
Peripheral blood smear:
Microcytic, hypochromic anemia
Hypochromic
Anisocytosis
(RDW= 18.6)
Poikilocytosis
38
Physiology of Iron
• Normal amount of body iron:
• Females 3g
• Males 4g
• Iron - 2.5g in Hgb or about 2/3 of total; some also in
myoglobin and other enzymes
• Principal storage (1 to 2g or 1/3 of total): Ferritin and
hemosiderin [stored in reticuloendothelial system
(RES) and liver]
39
Understanding iron metabolism:
• The body has no mechanism to excrete excess iron, therefore
absorption is strictly controlled
• Free iron is toxic therefore it is bound to proteins (transferrin,
apoferritin, or albumin)
• During iron deficiency, more transferrin is produced to carry extra iron
that is absorbed (increased “Total Iron Binding Capacity”)
• Some iron binds to apoferritin to form a water soluble storage and
transportable form called ferritin
• Excess iron is stored in bone marrow as water insoluble hemosiderin
40
Physiology of Iron
• Absorption:
• Duodenum/upper jejunum
• Heme absorbed intact (25% absorption rate)
• Inorganic iron (5% absorption rate)
• Facilitated by:
• Fe+++ (ferric) reduction (HCL) Fe++ (ferrous)
• Note: Ascorbic acid chelates Fe++
• Decreased by phytates & phosphates in vegetables, tannins
in tea & wine
• Absorbed Fe++ oxidized to Fe+++ and bound to
transferin. Complex internalized via cellular Tfn
receptor (TfR).
41
2+
42
Iron - Storage
• Iron is stored in liver, marrow, spleen
• Two major forms: hemosiderin and ferritin
• Hemosiderin
• Aggregates of Fe3+ core crystal stripped of apoferritin
• Water insoluble
43
Iron - Storage
• Ferritin
• Apoferritin (protein shell) + Fe3+ OH(3) (stored in core)
• Water soluble
• Readily available
storage form
44
Iron - Storage
45
Physiology of Iron
• Losses:
• 1 mg/day – mostly occult GI and skin
• Menstruation 30 mg/month
• Pregnancy – 1g to fetus
• Conditions that increase iron requirement
• Menstruation: 1-2 mg / day
• Pregnancy: 3-4 mg / day
• Blood loss
• Rapid growth
• Overload an issue; no excretory mechanisms:
• 1 unit PRBC = 200 mg of iron
46
Iron - Regulation
• Iron absorption at mucosal level regulated by the
amount of storage iron (ferritin and transferrin) in
mucosal cells →body stores
47
Low iron stores and no inflammation
2+
⇑Hepcidin 48
Trasferrin levels increase when
iron stores decline
• Laboratory:
Order one test to confirm diagnosis:
Ferritin = 5 µg/L [nl ~ 30-40]
51
Dx: Iron Deficiency Anemia
Key questions:
• Is there a source of increased loss of iron (e.g.. physiological or
pathological blood loss)?
• Is there sufficient iron in the diet (e.g., strict vegans)?
• Is iron absorption impaired (e.g. gastrectomy, small bowel
pathology, celiac disease)?
• Is there increased need for iron (e.g. “milk babies”)
52
Iron Deficiency
• Management
• Oral iron is drug of choice (ferrous sulphate, gluconate, or
fumarate)
• must be given in an adequate daily dose for a long enough
period of time
• Usual dose is 300 mg t.i.d. for 6 months.
• Improvement in symptoms and lab abnormalities occurs
within 2 weeks; iron stores replenished ~3 months after
hgb normalizes
53
B12 & Folic Acid Deficiency
Anemia
Case – Macrocytic Anemia
• 75 year man
• Presents with progressive dyspnea and unsteady gait
• Physical exam
• Pale, mild jaundice
• Decreased position sense
• Lab exam
• Hb 6.5 (low), MCV 125 (high)
• WBC 3.0, normal differential
• Platelets – 120
• Bilirubin mildly increased 35, LDH 950 ( normal < 180)
2
Hypersegmentation, macrocytes
3
Macrocytic Anemia
• Definition
• Anemia associated with MCV >97 fL.
• Classification (2 main groups)
• Megaloblastic vs normoblastic erythropoiesis
• Bone marrow morphology is the main way to distinguish between these 2 groups
• Megaloblasts = dyserythropoesis
• Hypersegmented neutrophils in megaloblastic anemia (due primarily to folate/B12
deficiency)
4
Megaloblastic Change
• Mechanism:
• Selective reduction in the rate of DNA synthesis
• Cytoplasmic components (RNA and protein, including
hemoglobin) are relatively increased
• Cytoplasmic maturation is faster than maturation of
nucleus = nuclear-cytoplasmic asynchrony.
• “Megaloblastic” change in red cell precursors =
dyserythropoiesis
5
Megaloblastic Change
• Many different cell types are affected.
• Affects all cells in rapid division
• Inhibits mitosis (cells arrested in S phase cell cycle)
• Most noticed in bone marrow but intestinal epithelial cells,
skin basal cells, etc are also affected
• The 2 main causes are vitamin B12 and folic acid
deficiency.
• Essential vitamins for DNA (purine) synthesis
6
Dyserythropoiesis
• Morphological abnormalities in erythroid precursors such
as:
• Macrocytosis
• immature chromatin pattern compared to cytoplasm =
nuclear:cytoplasmic asynchrony
• nuclear budding (early karyorrhexis)
• late cell division
• karyorrhexis
• May be “megaloblastic” change due to Vitamin B12 or folic
acid deficiency, OR *“megaloblastoid” change due to
myelodysplasia (a clonal process which may progress to
acute leukemia).
*Technically, this term should be used only
when B12/folate deficiency excluded
7
Normoblastic
Erythropoiesis
Pronormoblast
8
Megaloblastic
Erythropoiesis
9
)
10
Megaloblastic erythropoiesis is INEFFECTIVE
erythropoiesis
• Affected cells either die or omit the last few stages of normal
maturation
• Shortened life span of erythroid cells –
• intramedullary hemolysis
• Increased bilirubin (jaundice)
• Increased lactate dehydrogenase (LDH)
11
Vitamin B12
or Cobalamin
Source
• Bacterial synthesis (not made by plants or animals)
• Available to humans through animal, fish, eggs and dairy products,
nitrogen fixing bacteria in legumes
• Requirements - 2.4 µg/day
• Stores - 2-5 mg (supply 3-5 years) in liver
• Daily diet – 1-2 µg
12
13
14
Dietary B12 (cobolamine, Cbl)
released by peptic digestion →
bind to salivary protein
haptocorrin
Intrinsic Factor
(IF)
-Secreted by
gastric
parietal cells
Autoantibodies -Required for
in pernicious absorption of
anemia: B12
1. Block binding
of B12 to IF
2. Parietal cell
3. Prevent B12-
IF complex
binding to cubulin
Trancobolamin
e (TC)
transports Cbl
to tissues
15
Causes of B12 Deficiency
• Dietary :
• Rare
• More likely in strict vegans
• Intrinsic factor related
• Lack of IF due to destruction of parietal cells
• Autoantibodies to IF or receptors
• Lack of absorption
• Surgical resection of distal ileum
• Abnormal intestinal bacteria (blind loop syndrome)
• Competition by helminths
• Fish tapeworm (D. latum, seen in Finland) absorbs large quantities of intestinal B12
16
Actions of B12 and Folate:
• Folate is directly required for Purine (DNA) synthesis,
B12 is indirectly involved through folate metabolism
• Only tetra-hydro folate (THF) can participate in purine
synthesis
• Dietary folate is converted to THF and then to methyl-THF
• Methyl-THF can be converted back to THF if B12 is present
• Only B12 can transfer the methyl group from Methyl-THF to
homocysteine
• In the absence of B12, most folate is “trapped” as methyl-
THF , levels of THF decline, and DNA synthesis suffers
17
Physiologic Role of B12:
• Methyl-Cbl – In the Cytoplasm
• Adenosyl-Cbl – In the Mitochondria
18
Actions of B12 and Folate:
• Cytoplasmic action of B12: (Folate dependent)
• B12 is the coenzyme in a reaction which transfers the
methyl group from methyl- tetra-hydrofolate (THF) to
homocysteine (which is converted to methionine)
• THF is essential for purine (DNA) synthesis
• In the absence of B12, folate is “trapped” as methyl-THF
and DNA synthesis suffers
• Concomitant methionine deficiency may worsen internal
availability of folate
19
Actions of B12 and Folate:
• Mitochondrial action of B12: (Folate independent)
• Adenosyl-Cbl acts as coenzyme for conversion of
methylmalonyl-CoA to succinyl-CoA
• ? Associated with myelin formation
20
Anemia due to B12 or Folate Deficiency
• Folate administration will treat anemia due to either folate or B12
deficiency
• Neuropathy of B12 deficiency may be aggrevated by folate
• B12 administration will not correct anemia due to folate deficiency
21
CMAJ • August 3, 2004; 171 (3). 22
Folic Acid - Metabolism
• Folate cofactors essential for one carbon transfers
• Purine synthesis
23
Diagnosis – B12/Folic acid deficiency
• History:
• Dietary abnormality of 1 to 6 months’ duration
• Remember B12 stores last 3-5 years
• Folic acid - weeks
• Previous GI surgery, or malabsorption.
• History of autoimmune diseases
• Pernicious anemia: loss of IF production by parietal cells, a
common cause of B12 deficiency
• History of alcoholism, or other causes for malnutrition
• common cause of folic acid deficiency
• Ingestion of anticonvulsants (affects folate levels).
24
Diagnosis
• Physical Exam:
• Effects of anemia (pallor, edema, flow murmurs, congestive failure).
• Features of malabsorption (weight loss, surgical scars).
• Effects on other end-organs (epithelial atrophy, neurological involvement in
vitamin B12 deficiency only).
• Jaundice – intramedullary hemolysis
25
Diagnosis
• Investigation:
• Blood counts (anemia >leukopenia and thrombocytopenia).
• Peripheral blood smear (oval macrocytes, hypersegmented
neutrophils).
• Biochemical evidence of hemolysis ( bili, LDH).
• Bone marrow (megaloblastic maturation, erythroid
hyperplasia).
• Serum and RBC B12 and RBC folic acid levels.
• [Schilling test if indicated (test for B12 absorption)]
• Autoantibody work-up.
26
Peripheral blood and Marrow - B12/Folic Acid
Deficiency
Megaloblastic anemia
27
B12 Deficiency - Treatment
• Intramuscular B12 supplementation required since most causes are
due to malabsorption
• Hydroxycobalamine, 100 mg I.M. daily x 1-2 weeks to replenish stores
then monthly for life
• Expect rapid hematologic response (< 48hrs)
• Neurologic improvement slower, may not be complete
28
Folic Acid Deficiency - Treatment
• Oral supplementation, 1 mg/day for deficiency
29
Management
• Pregnancy (association of folate deficiency and neural tube defects).
• Active hemolysis (folate replacement).
• Patients having gastric or ileal resection should automatically go on
B12 injections post-op.
30
Thalassemia & sideroblastic
anemia
Case
• 59 y/o man of Mediterranean origin comes in for
routine check up
• Asymptomatic
• Physical exam is normal
• CBC performed as part of check up
• Hgb: 11.9 gm/dL
• MCV: 67 fl
• Platelets: 300,000 / cu mm
• WBC: 4,900/mm3
• Blood film: Microcytosis
WBC differential normal
• Reticulocyte: 80,000/ cu mm
2
Case 2-Peripheral Blood Smear
Target cells
Normal
3
Microcytic hypochromic anemia:
Etiologic differential diagnosis
• Iron deficiency anemia
• Thalassemia trait
• Anemia of chronic inflammation/disease
• Sideroblastic anemia
• Lead poisoning
4
Case Continued
5
Congenital Hemoglobin Abnormalities
• Affect over 400 million people worldwide
• Qualitative defects of globin chains: Structural hgb abnormalities (aka
hemoglobinopathies)
• Quantitative abnormality of Hgb production: Thalassemias
6
7
Genetics of globin
• 2 Alpha globin genes are on chromosome 16
(4 per diploid cell)
8
Human Globin Genes
Note: 2 α genes on each chromosome 16 = total 4 α genes
9
Human Globin Genes
Note: 2 α genes on each chromosome 11 = total 4 α genes
10
Thalassemia – Molecular basis
• The thalassemias are named after the affected globin chain – Alpha
thalassemia and Beta thalassemia
• α Thal: Caused by deletion of 1, 2, 3 or 4 α globin genes
• ß Thal: >100 different mutations, divided into ß0 and ß+ types
• ß0 – No ß chain produced - Disruption of RNA splice sites or nonsense mutations in exons
• ß+ - Reduced amount of ß chain produced - Introduction of new RNA splice sites or
promotor region mutations
11
Thalassemias –
Adult Hb = α2β2
Mechanisms of anemia Fetal Hb = α2γ2
Hb A2 = α2δ2
• Beta Thalassemias
• Decreased synthesis of β chains – compensatory increase
in γ and δ chains → increased levels of fetal Hb (α2γ2) and
Hb A2 (α2δ2)
• The unpaired excess α chains precipitate and severely
damage cell membranes
• RBC Precursors → Apoptosis in marrow (=ineffective
erythropoiesis) → reduced RBC and ↑ iron absorption
• Mature RBC → destroyed by splenic macrophages → markedly
reduced life span of RBC (= hemolytic anemia)
• Tetramers of α chains have very high O2 affinity → poor
delivery of oxygen to tissues
12
Thalassemias – Adult Hb = α2β2
Fetal Hb = α2γ2
Mechanisms of anemia …2 Hb A2 = α2δ2
• Alpha Thalassemias
• Alpha chains required for all hemoglobin types
• Complete absence of alpha chains is incompatible with normal
fetal development
• Tetramers of β chains (Hb H) and γ chains (Hb Bart) are
stable and only mildly toxic to cell membranes
• But they have very high O2 affinity – poor delivery of
oxygen to tissues
13
Molecular Basis of Thalassemias
β-Thalassemia α-Thalassemia
Number of 2 (1 per chromosome 11) 4 (2 per chromosome 16)
Globin Genes
Genetic Point mutations Gene deletions
abnormality
Molecular Either complete absence No transcription from
(β0) or reduced affected gene(s).
consequence transcription (β+).
15
Thalassemia – Pathologic Findings
• RBC are microcytic and hypochromic
Target Cell
Hypochromia
(increased
central pallor
Poikilocytosis
(variable shapes)
16
a-Thalassemia
• Clinical syndromes
• 4 syndromes described:
• silent carrier state
• a-thal trait
• HbBart (=γ4) and HbH (=β4) disease
• homozygous a-thal (hydrops fetalis)
• corresponds to 1 to 4 a-globin gene deletions
• ? protection against malaria and hgb S
17
a-Thalassemia
• Clinical History
• Onset in early childhood
• anemia due to hemolysis and ineffective erythropoiesis
• family history
• Physical Examination
• splenomegaly
18
Prevalence of Alpha Thalassemia
19
a-Thalassemia
• Investigation
• microcytosis, hypochromasia, target cells
• some RBCs may show precipitated b-globin (hgb H bodies) (hgb H disease)
• Molecular genetics: gene deletions
• Management
• genetic counseling
20
b-Thalassemia
• Features
• gene deletions uncommon; point mutations in promoter
region lead to reduced mRNA
• only 2 loci, thus clinical features are more distinct
• high prevalence in Mediterranean area
21
b-Thalassemia
• Diagnosis
• History
• family and ethnic history
- b-Thal Major (“Cooley’s anemia”)
• homozygous disease presents in early childhood (HbF ➔ HbA)
-debilitation, premature death
-failure to thrive
• Transfusion dependent
- b-Thal minor (heterozygous) and b-Thal intermedia often present as mild,
asymptomatic anemia
22
b-THALASSEMIA
• Physical Exam
• hepatosplenomegaly, abnormal facies (marrow expansion), bony
deformities
- b-Thal minor usually completely normal, often discovered
incidentally
23
β-Thalassemia Major
1. Severe hypochromic anemia – tissue hypoxia – stunted growth etc
2. Damage to RBC by excess α chains
a. Destruction in spleen – enlarged spleen
b. Compensatory increase in red cell production - marrow hyperplasia
c. Dependence on blood transfusions – iron overload - cirrhosis
26
Sideroblastic Anemias
• Features
• iron is present in normal amounts in erythroid precursors
• abnormally distributed in mitochondria
• unavailable for heme synthesis
• heterogeneous group of disorders
• X linked
• Drug or toxin mediated
• Myelodysplastic syndromes
27
Sideroblastic Anemias
• DIAGNOSIS
• History
• positive family history rarely (X Linked variety)
• ingestion of alcohol, drugs (anti-TB, prior alkylator chemotherapy)
• chronic lead poisoning (industrial exposure, ingestion)
• no specific features
28
Sideroblastic Anemias
• Physical Exam
• no severe effects from anemia
• splenomegaly is uncommon
• clinical manifestations of lead poisoning, chronic alcohol ingestion, TB, etc.
• Investigation
• mild to moderate anemia and microcytosis/hypochromasia
• pathognomonic ringed sideroblasts in marrow
• normal iron stores
29
Ringed Sideroblasts
30
Sideroblastic Anemias
• MANAGEMENT
• pyridoxine (vitamin B6) useful infrequently
• transfusion support may be needed sometimes
• bone marrow transplantation in rare patients
• removal of toxin: chelation therapy for lead, avoidance of chemicals
31
Laboratory Results in Microcytic Hypochromic
Anemias
Serum Fe TIBC % Serum Marrow Fe
Saturation ferritin
Iron ↓ ↑ ↓ ↓ ↓
Deficiency
Thalassemia N N N N ↑
Anemia of ↓ N-↓ ↓ N-↑ N-↑
Chr Dz
Sideroblastic ↑ ↓ ↑ ↑ ↑
anemia
32
Directed Learning Activity in
Hematology
Topic: Normocytic Anemias
1
Outline: Normocytic Anemias
• Reticulocyte: Normal physiology, importance in
classifying normocytic anemias
• Hemolytic anemias: Classification, abnormal
hemoglobins, red cell membrane abnormalities,
red cell enzyme abnormalities
– Hereditary spherocytosis and sickle cell disease
emphasized
• Autoimmune hemolytic anemia: Mechanism and
consequences
• Anemia of chronic inflammation:
Pathophysiology and pathological findings
2
Central pallor
4
Mutation affecting which of the following can
result in hemolytic anemia?
7
Mechanisms of increased Reticulocyte
Count in Anemia
• 1. Early release of immature reticulocytes (which
take longer to mature fully in blood)
– For every decrease of 5 units in hematocrit (1.5-2 g
Hgb), retics are released a day early
• 2. Increased amount of erythropoiesis in the
marrow
– In adults with reserve space in the long bones,
erythropoiesis can increase 6 to 8 fold normal levels;
retics can increase proportionately.
• These two mechanisms usually coexist
• Reticulocytes are slightly larger than RBCs
8
Normocytic Anemias with High Retic
Count– Increased Destruction
• Increased Destruction = Hemolytic anemias
– Next to iron deficiency anemia, this is the most
frequent type
– Many schemes of classification
– Most common causes worldwide are malaria,
hemoglobinopathies, and thalassemia.
– In North America, most patients have autoimmune
hemolysis
• NOTES:
– Acute blood loss also will lead to increased demand for RBC production and
therefore reticulocytosis.
– Most hemolytic anemias are Normocytic, may be slightly macrocytic when retic
count is high
9
Hemolytic Anemias
10
Classification of Hemolytic Anemias
• Etiologic:
– 1. Red cell abnormalities (Corpuscular defects)
– 2. Extra-corpuscular defects
• Intravascular vs Extravascular
– Intravascular lysis of RBC:
• Mechanical (artificial heart valve), physical (heat), or organic/
chemical (claustridial toxin, complement)
• Hemogobinemia, hemoglobinuria, hemosiderinuria
– Acute tubular necrosis in severe cases
• ↓↓↓Haptoglobin, ↑LDH, ↑bilirubin
– Exatrvascular lysis of RBC:
• Opsonized (complement) or rigid RBCs (spherocytes)
destroyed by macrophages in spleen (and liver)
• ↓Haptoblobin, ↑LDH, ↑bilirubin, gall stones
11
Classification of Hemolytic
Anemias: 1. Corpuscular Defects
• Hereditary
1. Membrane defect (spherocytosis, elliptocytosis, etc)
2. Enzyme defect
[Glucoze-6-Phosphate-Dehydrogenase (G6PD)
deficiency; Pyruvate Kinase (PK) deficiency)]
3. Hemoglobin abnormalities
(thalassemias, hemoglobinopathies)
NOTE: Thalassemias are microcytic/ hypochromic, but also have a
hemolytic component.
• Acquired
1. Membrane abnormality-paroxysmal nocturnal
hemoglobinuria (PNH)
12
Classification of Hemolytic Anemias:
2. Extracorpuscular Defects
A. Immune hemolytic anemias
1. Autoimmune hemolytic anemia
2. Transfusion of incompatible blood
B. Nonimmune hemolytic anemias
1. Chemicals
2. Bacterial infections, parasitic infections (malaria),
venoms
3. Hemolysis due to physical trauma to RBCs
- hemolytic - uremic syndrome (HUS)
- thrombotic thrombocytopenic purpura (TTP)
- prosthetic heart valves
4. Hypersplenism
13
Work Up in Suspected Case of
Hemolytic Anemia
• History:
– Family/ethnic history.
– Drug history.
– Presence of autoimmune diseases,
lymphoproliferative processes.
– Manifestations of anemia.
– Signs of destruction of RBC - heme converted
to bilirubin.
14
Diagnosis of haemolytic anemia
• Physical Examination
– Effects of anemia
– Jaundice/dark urine ( bilirubin)
– Splenomegaly (hypertrophy with clearance of RBC’s)
– Signs related to primary diagnosis
(autoimmune,lymphoproliferative disease)
• Notes:
– Reticulocytosis due to acute blood loss is NOT
accompanied by increased bilirubin or LDH.
– Anemia due to CHRONIC blood loss is usually
hypochromic/ microcytic because iron lost in the
blood leads to iron deficiency.
15
Tests to Determine Mechanism of
RBC Destruction-1
• Blood film and morphology
– Spherocytes (hereditary, immune destruction)
– Helmet or bite cells – (oxidative injury, enzymopathy)
– Fragments (mechanical destruction, intravascular
pathology)
– Malarial parasite
• Hemoglobinuria and decreased haptoglobin in
blood → intravascular hemolysis
• Osmotic fragility test
– Spherocytes are lysed at mildly hypotonic solutions
– Their spherical shape does not have any excess
membrane to allow increase in cytoplasmic volume
16
Tests to determine Mechanism of
RBC destruction-2
• Direct Antiglobulin Test (DAT/Coomb’s test)
– Antibody on surface of RBC
• Hgb electrophoresis if hemoglobinopathy
suspected (also sickle prep)
• Specific enzyme assay (G6PD, PK deficiencies)
• Molecular tests for thalassemia
• Family screening
17
Classification of Hemolytic
Anemias: 1. Corpuscular Defects
Membrane defects
18
Patient presentation: Hemolysis
• 30 year man
– History of anemia and recurrent episodes of
jaundice
– Father and brother had similar episodes and
gallstones at young age
• Exam – splenomegaly
• Lab – hgb 12.8
• Reticulocytes 280 x 103 (normal 20 -100 x 103)
• Bilirubin (total) 35 ( nl= 0.3-1.9); direct (conjugated)
8.0 (nl= 0-0.3)
• DAT (direct antiglobulin/Coomb’s test) - negative
19
Reticulocyte
21
Hereditray
Spherocytosis
Mutations affect
Autosomal
Dominant (75%)
or Autosomal
recessive (25%)
25
Classification of Hemolytic
Anemias: 1. Corpuscular Defects
Enzyme defects
26
RBC Enzymopathies
• Commonest are glucose-6-phosphate dehydrogenase
(G6PD) and pyruvate kinase (PK).
• G-6-PD Deficiency
– Enzyme required to regenerate NADPH and glutathione, which are
critical to avoid oxidative injury
G-6-PD Deficiency
– Affects about 10% of US black population.
• Also many in Africa, middle east, and south Asia – offers
protection against falciparum malaria
– Exposure to oxidative drugs (antimalarials, sulfa,
phenacetin, aspirin, vit K derivatives) or certain beans
precipitates attack of hemolysis
• Hemoglobin is oxidized and precipitates as Heinz
bodies.
• These older RBCs loose deformability
• Splenic macrophages “pluck out” the Heinz bodies → bite
cells
• Hemolysis is acute and of variable severity
– Females have two RBC populations due to random activation of X
chromosome and most females are clinically unaffected.
• Self-limited because only older RBCs are eliminated but
young ones are unaffected
28
G6PD Deficiency
29
Classification of Hemolytic
Anemias: 1. Corpuscular Defects
Hemoglobin abnormalities
30
Hemoglobinopathies:
• Hb S, C, E and D are the most prevalent
– Homozygous state = Disease
• Hemolytic anemia (severe in Hb SS, mild in Hb
CC)
• Microcytosis due to reduced Hb synthesis (E)
– Heterozygous state = Trait
• Denoted – AS, AE, AD etc. Or double
heterozygous state such as SC, Sickle-Thal, etc
• Offers some protection against falciparum malaria
(Sickle cell trait)
– 8% of African Americans are heterozygous for Hb S and
2.3% for HbC
• Can increase the severity of thalassemia (E, D)
31
Hemoglobinopathies (continued)
32
33
Sickle Cell Disease:
• Chronic hemolytic anemia characterized
by sickle-shaped red cells caused by
homozygous inheritance of Hemoglobin S
• Commonest hereditary anemia in the
world and in the US
– The sickle-cell gene occurs widely throughout
Africa and in countries with African immigrant
populations, some Mediterranean countries,
the Middle East, and parts of India
34
Sickle Cell Disease: Molecular Pathogenesis
35
Sickle Cell Anemia –
Laboratory Findings
• Anemia-normocytic or slightly
macrocytic
• Leukocytosis (chronic neutrophilia)
• Thrombocytosis-usually
mild<1000k/cmm
• Reticulocytosis
• Peripheral smear: few sickle shaped
red cells, polychromatophilia,
Nucleated RBC,
Howell-Jolly bodies
• Hb - electrophoresis
36
Hemoglobin Electrophoresis
At Alkaline pH At Acid pH
C C= A2, E C
S = D, G
37
Sickle Cell Anemia:
Clinical features
• Due to severe hemolytic anemia
– slow growth and development in children
– bilirubin stones
– congestive heart failure from chronic anemias
and cardiac overload compensation
38
Sickle Cell Anemia:
Clinical Features-2
Consequences of vaso-occlusion of the
microcirculation (tissue ischemia and
infarction)
– infarction of spleen, brain, marrow, kidney,
lung,
– aseptic necrosis of bone
– central nervous system and ophthalmic
vascular lesions
– many suffer from chronic pain, and end up as
suspected narcotic abusers
39
Splenic sequestration, Acute Pain, infarcts, ulcers,
lung syndrome, Priapism papillary necrosis
Occlusion of Vessels
Reduced elasticity
of red cells
Hemolysis
Anemia Jaundice
Stunted Increased
Growth Erythropoiesis
Parvovirus
Aplasia
40
Sickle Cell Anemia - Therapy
• Preventive measures:
– Infections (penicillin prophylaxis and
pneumococcal vaccination)
– Fever
– Dehydratation
– Acidosis
– Hypoxemia
– Cold exposure
• Blood transfusions for severe anemia
41
Sickle Cell Anemia: Treatment
•Allogeneic stem cell transplantation is only
curative method.
•New approaches to therapy
– Activation of hgb F synthesis: 5-azacytidine,
hydroxyurea; Experimental: inhibition of
BCL11A, a transcription factor that “silences”
gamma globin gene expression→increased
hgb F (J Clin Invest 2015. 125: 2363-2368; Blood, 2015.
126:89-93)
– Antisickling agents acting on hemoglobin or
membrane
42
Classification of Hemolytic Anemias:
2. Extracorpuscular Defects
43
Immune Hemolytic Anemias
44
Alloimmune Hemolytic Anemias
• Examples include:
– Hemolytic disease of the newborn: Rh- mother
sensitized. Anti-Rh IgG crosses placenta and
hemolysis of Rh+ RBC of fetus occurs.
– Immediate hemolytic transfusion reactions:
Mismatching for ABO, Kell, Fya etc. Pre-existing IgM
Abs bind transfused donor RBC and cause a C’-
mediated intravascular hemolysis.
– Delayed hemolytic transfusion reactions: Hemolysis
5-10 days after transfusion. Anamnestic response in
pre-sensitized patients. Initial Ab screen negative
due to low titers.
45
Autoimmune Hemolytic Anemia
(AIHA)
• Antibodies made to surface antigen(s) of
patient’s own (self) RBCs
• Two types defined by the temperature at
which the antibodies bind to RBCs
– Warm AIHA
– Cold AIHA
46
AIHA
Warm AIHA
abs bind at 37 C
usually IgG and not C’ binding
extravascular hemolysis
Cold AIHA
abs bind 4-30 C.
usually IgM and fix C’
usually Intravascular hemolysis.
Diagnostic feature of AIHA: Direct antibody test (DAT,
also called Direct Coomb’s test): Detects the presence
of IgG and/or C3 on patient’s RBC
(Indirect Coomb’s test : detects presence of antibody in
patient’s plasma)
47
Dx of AIHA: DAT
http://www.merck.com 48
AIHA: Etiology
• Idiopathic – most common
• Drugs
– quinidine, high-dose penicillin, alphamethyldopa –usually IgG ,
warm
– infections (EBV, mycoplasma) – IgM, cold
• Autoimmune disease
– e.g. systemic lupus erythematosis
• Lymphoid malignancies
• Symptoms and signs
– significant anemia
– sometimes jaundice
– splenomegaly
49
Cold AIHA
• RBC agglutination at 4 C, usually by IgM.
• Indirect Coomb’s test can determine titers and thermal
amplitude of responsible autoantibody.
• Antibody binding and C’ fixation <30C not clinically
significant
– Bound IgM fixes C’ at low temp but does not fully activate
compliment. At 37C IgM is released but C3b remains on RBC
surface → functions as an opsonin → extravascular hemolysis
by splenic macrophages
• Associated with infections (mycoplasma pneumoniae or
infectious mononucleosis); B cell lymphoid malignancies
50
AIHA
Warm Antibody Type
Primary Idiopathic
51
Autoimmune Hemolytic Anemias:
Salient Facts
52
AIHA: Management
53
Classification of Hemolytic Anemias:
2. Extracorpuscular Defects
54
Mechanical Hemolysis
• Related to mechanical prosthetic heart valves, valve
malfunction, and erythrocyte fragmentation syndromes.
• Diagnostic abnormality is the presence of fragmented
RBC (schistocytes).
• Most dangerous group is microangiopathic hemolytic
anemias (MAHA) -many are fatal.
– TTP, HUS, septicemia with/out DIC
– Pregnancy– related complications (Hemolysis, Elevated Liver
enzymes, Low Platelets: HEELP)
– malignant hypertension
• Treatment usually targets primary problem.
• Plasmapheresis is helpful for TTP, HUS.
55
Fragmented red celld (schistocytes)
56
Normocytic Anemias with
Decreased Red Cell Production
57
Normocytic Anemia: Normal or
Decreased Red Cell Production
58
Bone Marrow Pathology
• RBC production failure
– aplastic or hypoplastic anemia
– RBC precursors may be replaced (metastatic
CA, myeloma, leukemia, myelofibrosis, etc.)
– RBC precursors may be abnormal
(dyserythropoietic anemias) – usually
hereditary.
• There is almost always pancytopenia
(discussed later)
59
6
0
Aplastic Anemia
• Definition
– Pancytopenia with marrow hypocellularity
• Incidence
– Rare –
• 1-2 per million
• 5 -7 per million in Asia
• peaks in teens/twenties
6
1
Aplastic anemia
• Pathophysiology
– Immune or toxic injury to stem cell
– Marrow stroma and growth factors usually
normal
• Diagnosis
– Marrow aspirate and biopsy →replaced by fat
– Cytogenetics
– Chromsome breakage in Fanconi’s anemia
6
3
Aplastic anemia
• Prognosis
– Severe AA: retics < 20,000/cmm, neuts
<0.5, platelets <20
– 2/3 criteria: 20% mortality at 1 year
• Therapy
– Supportive – transfusion
– Immunosuppressives (ATG, cyclosporin)
– Allogeneic stem cell transplant
Normal bone
marrow biopsy
Aplastic anemia
64
6
5
Case 1 – Pancytopenia
• 25 year old man, previously healthy
• Complains of general fatigue and bruising for
weeks
• Presents to ER with fever and “sensation of his
heart pounding in head”
– On exam
• Pale, p 130, t 39 C, RR 24
• Petechiae, no organomegaly
– CBC
– Leuk 1.0 ( Neut 0.5, lymphs 0.4, mono 0.1) [ normal 4 to
10]
– Hgb 55 g/L (135 to 170)
– Platelets less than 10 ( normal 150 -400)
6
6
• Decreased Production
– Aplasia
– Dysplasia ( B12 /Folate)
– Infiltration (leukemia, lymphoma, myeloma, fibrosis,
metastses)
• Sequestration
– (hypersplenism)
• Increased Destruction
– Immune
– Non-immune
6
7
• Cellular marrow
– infiltrated or replaced ( e.g.,metastatic
CA, myeloma, leukemia,
myelofibrosis).
– Hematopoietic cells are abnormal:
myelodysplasia [stem cell defect that
leads to dysplastic maturation and
6
8
DDX: Pancytopenia
• Physical signs: non-specific
– Signs of bone marrow failure
• Pallor, bleeding, infection
– Signs of organ infiltration
• Hepatoslenomegaly
• Enlarged lymph nodes
• Gum hypertrophy
Petechiae
6
9
70
All statements about the condition seen in peripheral blood
smear below are correct except:
72
Case 3: Additional Tests
• ESR: 80 mm/hr (high)
• BUN: 42 (high)
• Creatinine 2.0 (high)
• Anti Nuclear Antibody 1:1256 (high)
• Complement C3/C4 Low
• Anti-ds DNA Positive
• Diagnosis
– Systemic Lupus Erythematosus (SLE)
– Renal insufficiency
– Anemia of chronic inflammation
• Possibly worsened by low erythropoietin
73
Anemia of Chronic Disease
(chronic inflammation)
• Features
– Mild-to-moderate anemia, variable-to-no
microcytosis
– hypochromasia, normal iron stores
– presence of chronic inflammatory disease or
neoplasm
74
Anemia of Chronic Disease
(chronic inflammation)
• Mechanism
– Inflammatory mediators (TNF, IL-1,-2)
• iron sequestered by acute phase reactant storage
proteins in RE system
• increased hepcidin → decreased iron release from
RE cells
• iron is sequestered in RE cells and poorly
mobilized into erythrocytes
75
Pathophysiology of ACD/I
76
Weiss & Goodnough NEJM 2005
Anemia of Chronic Disease
(chronic inflammation)
• Diagnosis
– History
• presence of significant other medical problems,
especially inflammatory diseases; absence of
history of increased iron loss
– Physical Exam
• little or no evidence of end-organ impairment from
anemia
• evidence of infection, inflammation, or neoplasm
77
Anemia of Chronic Disease/
Inflammation
• Investigations
• mild anemia, normocytic-to-mild microcytosis
(MCV rarely <75)
• ferritin is high-normal or increased
• Serum iron may be low but TIBC is normal
• stainable iron stores are increased on marrow
exam, but confined to macrophages
• Management
• aimed at primary disease
• seldom requires transfusion
• erythropoietin in severe cases
78
Anemia of Chronic Disease:
Experimental Treatments
79
Normocytic anemias: No Bone Marrow
Pathology
• Heterogeneous group consisting of
metabolic disorders resulting in decreased
RBC production.
– failure of EPO production – renal failure
– hypothyroidism (decreased response to and
decreased production of EPO)
– anemia of chronic disease/inflammation
(inflammatory mediators decrease iron
availability, antagonize EPO effect)
– combination of iron/B12/Folate depletion.
80
Erythropoietin (EPO)
81
Erythropoietin (EPO)
• Normally, inverse relationship between
hemoglobin and EPO levels
• Increased EPO in anemia
• Exogenous administration of EPO can
boost hemoglobin levels.
82
Erythropoietin (EPO) Therapy in
Anemia
• Chronic renal failure (EPO deficient state)
– replacement of hormone
• Anemia in patients with AIDS on AZT
• Anemia associated with chemotherapy for
malignancies
• Surgery to avoid blood transfusion (boost
preoperative hemoglobin)
83
Directed Learning Activity in
Hematology
Topic: Thrombocytopenias
Platelets
• Smallest cellular component of blood
• Circulate in an inactivated form
• Activated on contact with damaged blood vessel wall
(endothelial cell) and by other substances at site of injury
(collagen, ADP, etc)
• Form primary hemostatic plug; formed quickly but unstable
if not fortified by fibrin
• Initiate coagulation cascade providing phospholipid
leading to formation of fibrin clot
2
Platelets
• Megakaryocyte maturation and platelet production
stimulated by thrombopoietin
• Platelet lifespan- 7 to 10 days
• Clinical correlations
• Reduced platelet count → bleeding
• Too many platelets → risk of thrombosis
• Abnormally functioning platelets → bleeding or
thrombosis
3
Megakaryoblast
4
5
6
Thrombocytopenia
Definition:
• Mild: 80 - 150 x 109/L
• No problem expected with or without trauma or surgery
• Moderate: 20 - 80 x 109/L
• May bruise with mild trauma but usually not spontaneously; excessive bleeding is likely to
occur at surgery
• Severe: < 20 x 109/L
• Risk of spontaneous bruising and bleeding, which may be life threatening (e.g., intracranial
hemorrhage)
7
Classification of Thrombocytopenia
• Decreased production
• Increased destruction
• Sequestration*
9
Increased Destruction
• Immune
• idiopathic
• secondary:
• SLE, CLL, lymphoma
• Drugs, e.g., heparin
• viral: HIV
• Non-immune
• TTP/HUS
• DIC
• Sepsis
10
Sequestration
• Hypersplenism
• Normally 20% of platelets sequestered in spleen. In conditions of enlarged spleen, up to 80%
may be sequestered
• Platelet count usually >50x109
• Cardiopulmonary bypass
11
Approach to Thrombocytopenia
12
Approach to Thrombocytopenia
13
Approach to
Thrombocytopenia
5. Exclude possibility of TTP/HUS and DIC
• peripheral blood smear
• INR/PTT, fibrinogen, LDH, creatinine
14
Principles of Management
• Avoidance of aggravating factors
• Avoid medications which affect platelet function
• Intramuscular injections should be avoided in all patients with thrombocytopenia
• Investigate and treat underlying cause
• Platelet transfusion
• May “feed the fire” in immune mediated diseases
• Use single donor platelets when possible to avoid sensitization to multiple
antigens
15
Case 1 - Thrombocytopenia
16
Petechiae
•Pinhead size (1-3mm)
•Red; non-palpable
•Non-blanching; dependent areas
•RBC extravasation from capillaries
Pupura
•Confluent petechiae
•Purplish
•Wet or dry
17
Immune (or Idiopathic) Thrombocytopenic
Purpura (ITP)
• Diagnosis of exclusion
18
ITP
Can occur in association with:
• viral/bacterial infections
• autoimmune disease
• drugs
• lymphoproliferative disorders
• post transfusion
• idiopathic
19
Clinical Manifestations
• Mild cases may be asymptomatic
• No splenomegaly
20
Acute ITP
• 80% children, 20% adults
• M:F= 1:1
• Duration < 6 months
• Abrupt onset
• Preceding viral infection 2-21 days
• Spontaneous remission 1-2 months
21
Acute ITP--Treatment
• Treat if symptomatic, i.e., bleeding
Children:
• IVIG to avoid effects of steroids
Adults
• Prednisone 1-2 mg/kg
22
Chronic ITP
• 80% adults, 20% children
• M:F= 1:3
• Insidious onset
• Rarely resolves spontaneously
• Association: SLE, lymphoproliferative disorders
23
Chronic ITP-Treatment
• Immunosuppression:
• Prednisone-first line therapy
If patients fails to respond:
• Eliminate antibody producing B cells with monoclonal antibodies – rituximab (anti-CD20
antibody)
• Saturate Fc receptors on splenic macrophages- the site of immune destruction of
platelets
• Rh immune globulin
• IVIG
24
Chronic ITP-Treatment-2
• Thrombopoietin analogs
• Splenectomy-considered if prednisone failure or intolerant
• Platelet transfusion-used only in life-threatening hemorrhage because
of the extremely short life span of transfused platelets
25
HIT/TTP/HUS/DIC
Case: Thrombocytopenia
• 78 yo patient is admitted and treated for pneumonia with IV
antibiotics. Initial platelet count = 550.
27
Case: Thrombocytopenia
• What is the diagnosis?
28
Case: Thrombocytopenia
29
Dx:Heparin-Induced Thrombocytopenia (HIT)
30
HIT
• Antibody to heparin which has an affinity for Fc
receptors (PF 4) on platelets
• Result in platelet activation and consumption
• Associated with arterial or venous thrombi
• Need substitution of another anticoagulant
31
Heparin-induced thrombocytopenia
33
Case: Thrombocytopenia
• Findings on P/E?
• Further investigations?
34
Blood smear
35
TTP/HUS
TTP = Thrombotic Thrombocytopenic Purpura
HUS = Hemolytic Uremic Syndrome
• Two related conditions featuring fever, hemolysis,
thrombocytopenia, neurological symptoms and renal
insufficiency
• Causes:
• E. coli 0157-H7 serotype, HIV, medications
• Idiopathic
• Pathophysiology of TTP: vWF and ADAMTS13
• ADAMTS13 is a zinc-containing metalloproteinase enzyme that
cleaves large von Willebrand factor (vWf) multimers
[A Disintegrin And Metalloproteinase with ThromboSpondin motifs
13]
36
TTP - Mechanism
37
1. Platelets are consumed in the intravascular clots →
thrombocytopenia
2. RBC are injured by the fibrin strands in many
microvessels →microangiopathic hemolytic anemia
38
TTP/HUS
Investigations:
• CBC: anemia, thrombocytopenia
• Blood smear: RBC fragments (schistocytes),
thrombocytopenia
• BUN/creatinine: due to renal failure
• INR/PTT: normal
Therapy:
• Plasmapheresis/plasma infusiion
• Platelet-delivered recombinant ADAMTS13 in a murine
model prevents TTP(Pickens et al. 2015. Blood 21:3326-3334) Applicable to
humans?
39
TTP/HUS
HUS (Hemolytic Uremic Syndrome)
• Hemolysis, renal failure, thrombocytopenia
• More common in children
• May follow diarrheal illness (Escherichia coli O157:H7)
• Therapy same as TTP
40
Case: Thrombocytopenia
• 27 yo man is admitted with acute onset headache, fever, and
confusion
• Exam
• Febrile, tachycardic, hypotensive, confused
• Petichial rash
• Neck stiffness
• Lab
• WBC 25, neutophilia, Hb 140, platelet count =18
• INR 2.5, PTT 65 (both elevated), fibrinogen 0.5 (low)
• Why is the patient thrombocytopenic?
41
42
Disseminated Intravascular
Coagulopathy (DIC)
• Widespread intravascular deposition of fibrin with
consumption of coagulation factors and platelets
with accelerated fibrinolysis
• Search for underlying cause
• Malignancy: mucin secreting adenocarcinoma,
leukemia, especially acute promyelocytic leukemia (
FAB M3)
• Sepsis
• Obstetrical accidents
• Trauma, widespread tissue injury
43
DIC
Investigations:
• CBC: anemia, thrombocytopenia
• Blood film: RBC fragments, thrombocytopenia
• INR/PTT: prolonged due to consumptive
coagulopathy
• Fibrinogen: decreased
• Additional tests?
44
45
DIC
Treatment:
• Supportive care with blood products
• Identify and treat underlying cause
46
Laboratory Findings of Leukemias/Lymphomas (From Robbins)
Acute leukemia: The diagnosis of acute leukemia is by identification of blast forms in the peripheral blood and the bone
marrow. The white cell count is variable (either > 100,000 cells/µL or sometimes, < 10,000 cells/µL. Anemia is almost always
present. Platelet count is usually below 100,000 platelets/µL. Neutropenia is also a common finding in the peripheral blood.
Morphology varies based on lymphoblasts or myeloblasts. The nuclei of lymphoblasts in Wright-Giemsa-stained
preparations have somewhat coarse and clumped chromatin and one or two nucleoli; myeloblasts tend to have finer
chromatin and more cytoplasm, which may contain granules. The cytoplasm of lymphoblasts often contains large
aggregates of periodic acid-Schiff-positive material, whereas myeloblasts are often peroxidase positive.
Table. Characteristics of Common Lymphoid Leukemias, Non-Hodgkin Lymphomas (Modified from Robbins)
Name of the clinical Frequency Salient Morphology Immunophenotype Comments
Entity
Precursor B-cell 85% of childhood Lymphoblasts with TdT+, immature B cell Presents as acute
lymphoblastic acute leukemia irregular nuclear markers (CD19+, leukemia; less
leukemia/lymphoma contours, condensed variable expression of common in adults.
chromatin, small other B-cell markers)
nucleoli, and scant
agranular cytoplasm
Small lymphocytic 3% to 4% of adult Small resting CD5+, mature B-cell Involves nodes,
lymphoma/chronic lymphomas; 30% of all lymphocytes mixed (CD20+) marrow, and spleen;
lymphocytic leukemia leukemias with variable numbers most patients have
of large activated peripheral blood
cells; lymph nodes involvement
diffusely effaced
1
Follicular lymphoma 40% of adult Frequent small CD10+, BCL2+ & Involves nodes,
lymphomas "cleaved" cells mixed mature B cells marrow, and spleen;
with large cells; (CD20+) associated with
growth pattern is t(14;18).
usually nodular
(follicular pattern)
Mantle cell lymphoma 6% of adult Small to intermediate- CD5+, mature B cells Involves nodes,
lymphomas sized irregular (CD20+) that express marrow, spleen and
lymphocytes growing cyclin D1 GI tract; associated
in a diffuse pattern with t(11;14).
Diffuse large B-cell 40% to 50% of adult Variable; most Mature B cells Often arise at
lymphoma lymphomas resemble large (CD20+) with variable extranodal sites;
germinal center B expression of CD10, aggressive
cells; diffuse growth BCL2
pattern
Burkitt lymphoma <1% of lymphomas Intermediate-sized CD10+, CD20+ B cells Associated with
lymphoid cells with and BCL6+ t(8;14) and EBV;
several nucleoli; Endemic in Africa,
diffuse growth pattern; sporadic elsewhere;
frequent apoptotic increased frequency
cells (“starry-sky” in immunosuppressed
appearance) patients;
predominantly affects
children; often
presents with visceral
involvement; highly
aggressive.
TdT = Terminal deoxynucleotidyl transferase, an enzyme specifically expressed in pre-B and pre-T cells.