PATHOPHYSIOLOGY

OF ANEMIAS
 ANEMIA - DEFINITION

REDUCTION OF HEMOGLOBIN
CONCENTRATION BELOW
REFERENCE VALUE

• Anemia, like a fever, is a symptom of

disease that requires investigation to
determine the underlying etiology.
 ANEMIA – DEFINITION – cont.
• Anemia is strictly defined as a decrease in red blood cell
(RBC) mass.
• Methods for measuring RBC mass are time consuming,
are expensive, and usually require transfusion of
radiolabeled erythrocytes. Thus, in practice, anemia is
usually discovered and quantified by measurement of
the RBC count, hemoglobin (Hb) concentration, and
hematocrit (Hct).
• These values should be interpreted cautiously because
they are concentrations affected by changes in plasma
volume. For example, dehydration elevates these
values, and increased plasma volume in pregnancy can
diminish them without affecting the RBC mass.
 BLOOD PARAMETERS
• Hemoglobin concentration (Hg)
• F: 7,2 –10; M: 7,8-11,3 mmol Fe/l (12-18 g/dl)
• Erythrocytes count (RBC)
• F: 4-5,5; M: 4,5-6 x1012/l (4-6 x106 /µl)
• Hematocrit (Hct)
• F: 37-47; M: 40-54; (37-54%)
• Platelet count (Plt)
• 150 – 450 x 103/µl (150-450 x 109/l)
• Leukocytes count (WBC)
• 4-10 x 109/l (4-10 x 103/ µl)
 Erythrocytes parameters
– Mean corpuscular volume (MCV)
– N: 80-100 fl
– RDW (Red cell Distrubution Width)
– Higher RDW values indicate greater variation in size
– Mean corpuscular hemoglobin (MCH)
– N: 27-34 pg
– Mean corpuscular hemoglobin concentration
(MCHC)
– N: 310 – 370 g/l RBC (31-37 g/dl)
 Reticulocyte count
• Reticulocyte count = % immature RBC
• Normal 0.5-1.5%
• <0,5% Inadequate production
• >1,5% Increased production
 Classification of Anemia
I. Etiologic Classification
1. Impaired RBC production
2. Excessive destruction
3. Blood loss

II. Morphologic Classification

1. Macrocytic anemia
2. Microcytic hypochromic anemia
3. Normochromic normocytic anemia
 Impaired RBC Production
1. Abnormal bone marrow
1.1 Aplastic anemia
1.2 Myelophthisis : Myelofibrosis, Leukemia,Metastases

2. Essential factors deficiency

2.1 Deficiency anemia : Fe, Vit. B12, Folic acid, etc
2.2 Anemia in renal disease : Erythropoietin deficiency

3. Stimulation factor deficiency

3.1 Anemia in chronic disease
3.2 Anemia in hypopituitarism
3.3 Anemia in hypothyroidism
 Excessive Destruction of RBC
Hemolytic anemia
1. Intracorpuscular defect
1.1 Membrane : Hereditary spherocytosis
Hereditary ovalocytosis, etc.

1.2 Enzyme : G-6PD deficiency,etc.

1.3 Hemoglobin : thalassemia,

hemoglobinopathies
Excessive Destruction of RBC
2. Extracorpuscular defect
2.1 Mechanical :MAHA (Microangiopathic HA)
2.2 Chemical/Physical
2.3 Infection : Clostridium tetani
2.4 Antibodies : Haemolytic reaction, SLE
2.5 Hypersplenism
 Blood Loss

1. Acute blood loss : Accident, GI bleeding

2. Chronic blood loss : Hypermenorrhea
Parasitic infestation
 Morphological Classifications of
Anemia
• Microcytic- RBC volume
< 80 fl oz. (small RBC’s)
• Normocytic- RBC volume
80-99 fl oz. (normal RBC’s)
• Macrocytic- Blood volume <
100 fl oz. (Large RBC’s)
 Macrocytic Anemia
MCV > 94
MCHC > 31
1. Megaloblastic dyspoiesis
1.1 Vit. B12 deficiency : Pernicious anemia
1.2 Folic acid deficiency : Nutritional megaloblastic
anemia, Sprue, Other malabsorption
1.3 Inborn errors of metabolism : Orotic aciduria,etc.
1.4 Abnormal DNA synthesis : Chemotherapy,
Anticonvulsant, Oral contraceptives
 Microcytic Hypochromic
Anemia
MCV < 80
MCHC < 31
1. Fe deficiency anemia : Chronic blood loss, inadequate
diet, Malabsorption, Increased demand, etc.
2. Abnormal globin synthesis : Thalassemia with or without
Hemoglobinopathies
3. Abnormal porphyrin and heme synthesis : Pyridoxine
responsive anemia, etc.
4. Other abnormal Fe metabolism
 Normocytic Normochromic
Anemia
MCV 82 - 92
MCHC > 30
1. Blood loss
2. Increased plasma volume : Pregnancy, Overhydration
3. Hemolytic anemia : depend on each cause
4. Hypoplastic marrow : Aplastic anemia, RBC aplasia
5. Infiltrate BM : Leukemia, Multiple myeloma, Myelofibrosis, etc.
6. Abnormal endocrine : Hypothyroidism, Adrenal insufficiency,
etc.
7. Kidney disease / Liver disease / Cirrhosis
 Anemia clinical manifestation
• GENERAL ANEMIA’S SYMPTOMS:
– FATIGABILITY
– DIZZINES
– HEADACHE
– SCOTOMAS
– IRRITABILITY
– PALPITATION
– CAD, CHF
 Case 1
• A 1year-old infant presents at a clinic with lassitude, poor
muscle tone and delayed motor development.
• The mother is a known IV drug user and has two older
children who are in the custody of the state social services
agency.
• VS: tachycardia; tachypnea.
• PE: pallor; partial alopecia; ulceration of skin at corners of
mouth; smooth tongue; nails break easily and are spoon
shaped
• Labs: abnormally small and pale RBCs
• RBCs of different sizes and different shapes
• Decreased serum iron; increased total iron-binding capacity
and reduced percentage saturation;
• Micro Pathology of bone marrow: Erythroid hyperplasia with
decreased bone marrow iron stores on Prussian blue
staining
IRON DEFICIENCY ANEMIA
CHARACTERISTIC SYMPTOMS

– GLOSSITIS, STOMATITIS
– DYSPHAGIA ( Plummer-Vinson syndrome)
– ATROPHIC GASTRITIS
– DRY, PALE SKIN
– SPOON SHAPED NAILS
– BLUE SCLERAE
– HAIR LOSS
– PICA (APETITE FOR NON FOOD SUBSTANCES SUCH
AS AN ICE, CLAY)
– SPLENOMEGALY (10%)
– INCREASED PLATELET COUNT
 IRON DEFICIENCY ANEMIA
• MCV - ↓

• MCH - ↓

• MCHC - ↓ or N

• Fe - ↓

• TIBC - ↑

• TRANSFERIN SATURATION - ↓

• FERRITIN - ↓
 BLOOD AND
BONE MARROW SMEAR
• BLOOD:
– microcytosis, hipochromia, anulocytes, anisocytosis
poikilocytosis
• BONE MARROW
– high cellularity
– mild to moderate erythroid hyperplasia (25-35%; N 16
– 18%)
– polychromatic and pyknotic cytoplasm of
erythroblasts is vacuolated and irregular in outline
(micronormoblastic erythropoiesis)
– absence of stainable iron
IRON DEFICIENCY ANEMIA BLOOD SMEAR
IRON METABOLISM
The total body iron in a 70-kg
man is about 4 g.

•ABSORPTION IN
DUODENUM

•TRANSFERRIN
TRANSPORTS IRON TO
THE CELLS

•FERRITIN AND
HEMOSYDERIN STORE
IRON

•10% of daily iron is absorbed

 Iron deficiency pathophysiology
• Iron is vital for all living organisms because it is essential for
multiple metabolic processes, including oxygen transport, DNA
synthesis, and electron transport.
• Dietary iron is available in two forms: heme iron, which is found
in meat; and non-heme iron, which is found in plant and dairy
foods.
• Absorption of heme iron is minimally affected by dietary factors,
whereas non-heme iron makes up the bulk of consumed iron.
• The bioavailability of non-heme iron requires acid digestion and
depends on the concentration of enhancers (e.g., ascorbate,
meat) and inhibitors (e.g., calcium, fiber, tea, coffee, wine)
found in the diet.
• In states of overload, absorption decreases. Absorption can
increase three- to fivefold in states of depletion.
• Iron deficiency results when iron demand by the body is not met
by iron absorption from the diet.
 IRON DEFICIENCY ANEMIA
• ETIOLOGY:
• CHRONIC BLEEDING
» MENORRHAGIA
» PEPTIC ULCER
» STOMACH CANCER
» ULCERATIVE COLITIS
» INTESTINAL CANCER
» HAEMORRHOIDS
• DECREASED IRON INTAKE
• INCREASED IRON REQUIRMENT (JUVENILE
AGE, PREGNANCY, LACTATION)
 Case 2
• A 73-year-old Caucasian male with a negative
past medical history, who never was followed by
a doctor, is admitted to the hospital with a chief
complaint of constipation for 3 months.
• He also complains of profound weakness and
exertional chest pain and shortness of breath for
that last 3-4 weeks.
• He used to have daily bowel movements but
starting 3 months ago he started to complain of
severe constipation, passing large amount of
hard stool
• He lost 5 kg during last 3 months
 Case 2
• The rectal examination showed fresh
blood positive stools and large lobulated
mass, measuring around 11 cm with
multiple bleeding vessels
• Pathological examination of bioptic
specimen confirmed the rectal carcinoma
 Case 3
• A 35 year old woman is seen for easy fatigue for many
months.
• The patient had been well until a few months earlier, when
her menses, which had been profuse in recent years,
became even heavier.
• Five days before admission, she began to menstruate
profusely and had orthostatic weakness. She does not see
any obstetrician and does not take any vitamins.
• Lately, she has developed a taste for eating ice.
• She has no other complaint.
• Physical examination is positive for pale conjunctiva, and a
systolic murmur at left lower sternal border.
• Stools are negative for occult blood.
 Case 3
• Labs: Complete blood count - Hg 7.1
gm/dl, Hct 23%,
• WBC 5,400/mm3 (differential is normal),
• platelets 450,000/mm3;
• Mean Corpuscular volume (MCV) is 74 fl
(normal 85-95 fl);
• Red cell Distribution Width (RDW) is
17.1% (normal 13-15).
 SIDEROBLASTIC ANEMIAS
• Hereditary disorders (rare) or acquired conditions
• Synonim for MDS
• Defects involving incorporation of iron into the heme
molecule
• Higher SIDEROBLASTS number in bone marrow
• On a marrow stained with Prussian blue, a sideroblast is
an erythroblast that has stainable deposits of iron in
cytoplasm.
• When abundant, these deposits form a ring around the
nucleus, and the cells become ring sideroblasts
• Under normal circumstances, this iron would have been
used to make heme.
Congenital sideroblastic anemias

• Pyridoxine-responsive type
– Point mutations on the X chromosome have been identified that
result in a δ-amino levulinic acid synthase (ALAS-2) with very
low enzymatic activity
– This impairs the first crucial step in the heme synthesis pathway,
the formation of δ-amino levulinic acid,

• Pyridoxine-resistant, X-linked sideroblastic anemia

– The ABC7 gene mutation
– ABC-7 is an adenosine triphosphate (ATP)-dependent
transporter protein involved in the cytosolic transfer of iron-sulfur
complexes.
 Acquired sideroblastic anemias
Refractory anemia with ring sideroblasts (RARS)
– Myelodysplastic syndrome characterized by an
anemia in which at least 15% of bone marrow
erythroblasts are ringed sideroblasts,
– Approximately 15% of patients also have
thrombocytosis and 5% may also be considered a
form of the myeloproliferative syndrome (MPS).
– 3 to 12 % of patients with RARS progress to
leukemia.
– Interestingly, the more numerous the sideroblasts, the
lower the risk of progression
 Acquired sideroblastic anemias
• Copper deficiency ,
– a part of malabsorption syndrome or due to excessive zinc
intake (supplements)
– myelodysplastic syndrome with sideroblastic anemia and
leukopenia.

• Vitamin B-6 (pyridoxine) deficiency

– coenzyme in the first, rate-limiting step in heme formation
catalyzed by δ-ALAS

• Lead poisoning
– cause sideroblastic anemia by inhibiting several enzymes
involved in heme synthesis, including δ-aminolevulinate
dehydratase, coproporphyrin oxidase, and ferrochelatase.
 Case 4
• A 58-year-old black female complains of
weakness dizziness, anorexia, nausea, and
occasional vomiting over the past 3 months.
• She has also experienced shortness of breath
as well as numbness and tingling in the
extremities
• PE: beefy-red tongue; loss of balance, vibratory,
and position sense in both lower extremities
• Gastroscopy: achlorhydria (no hydrochloric acid
in gastric juice); atrophic mucosa, loss of rugal
folds in stomach
Labs: macrocytic, hypochromic anemia (MCV > 100); leukopenia (4,000) with
hypersegmented neutrophils; thrombocytopenia
Axial T2 Image of the
cervical spine reveals
intramedullary
hyperintensity in the
cervical cord involving
the posterior and the
lateral columns.
 Megaloblastic Anemia
• Anemia with macrocytic red cells (MCV > 100 fL)
• Low-normal absolute reticulocyte count
• BM shows intense erythroid hyperplasia with
abnormal morphology.
• Macroovalocytes and occasional megaloblasts
can be seen.
• Hypersegmented PMN
• A result of impaired DNA synthesis due to def. in
Folate and/or vitamin B12 (cobalamin).
 Pernicious Anemia
• Epidemiology:
– Most common cause of vitamin B12 def.
– About 2% of people over 60 have undiagnosed
pernicious anemia
– Most common in whites of Northern European
ancestry.
– Average age of diagnosis is approx 60.
B-12 Physiology
Normal B-12 absorption:
•Dietary B-12 binds to R factor
in saliva and gastric juices.

•In duodenum, pancreatic

enzymes promote dissociation
from R factor and binding to
Intrinsic Factor (IF)

•IF-B12 complex taken up by

ileal receptor cubilin.

•Released into plasma bound to

transcobalamines TC I, II, or III.

•Enters cells through receptor

mediated endocytosis and
metabolized into two
coenzymes: adenosyl-Cbl and
methyl-Cbl.
 Pernicious Anemia
• Pernicious anemia is a chronic illness caused by
impaired absorption of vitamin B-12 because of a lack of
intrinsic factor (IF) in gastric secretions.
• Pernicious anemia occurs as a relatively common adult
form of anemia that is associated with gastric atrophy
and a loss of IF production
• Pernicious anemia probably is an autoimmune disorder
with a genetic predisposition.
• Antiparietal cell antibodies occur in 90% of patients with
pernicious anemia but in only 5% of healthy adults.
Similarly, binding and blocking antibodies to IF are found
in most patients with pernicious anemia.
 Pernicious Anemia
• Pathophysiology
– Autoantibody to IF
• Two types:
– Type I blocks attachment of B-12 to IF
– Type II blocks B-12-IF complex to ileal receptor
– Present in up to 70% of patients with P.A.

– Autoantibody to gastric parietal cells

– Directed against the H/K-ATPase on cell membrane
– Leads to decline in number of parietal cells and IF production
– Leads to chronic atrophic gastritis and gastric atrophy.
– Found in 90% of patients with pernicious anemia.
 Symptoms of Cobalamin deficiency
• Megaloblastic Anemia.
• Gastrointestinal findings: Approximately 50% of patients have
a smooth tongue with loss of papillae. The tongue may be
painful and beefy red.
• Chronic atrophic gastritis with Achlorhydria (lack of HCl in
gastric juice)
• Elevated serum bilirubin reflective of increased RBC
breakdown due to ineffective erythropiesis.
• Nervous system: the most common symptoms are
paresthesias, weakness, clumsiness, and an unsteady gait.
• These neurological symptoms are due to myelin degeneration
and loss of nerve fibers in the dorsal and lateral columns of
the spinal cord and cerebral cortex.
Folate/B12 DNA Synthesis
 Case 5
• A 46 year old man, a vegetarian (on very restrictive diet),
presented with acute onset of paresthesia (“pins and needles”
sensation) involving both hands and feet of 15 days duration.
• He also complained of difficulty in walking and inability to feel
the ground for the same period.
• There was no history of trauma, fever, vomiting, diarrhea, upper
respiratory tract infection, visual disturbances, and bladder or
bowel incontinence.
• On examination, the patient was restless and agitated but his
higher mental functions were normal.
• There was impairment of sensation of fine touch, pin prick, joint
position and vibration in both hands and feet bilaterally.
 Case 5
• The initial laboratory investigations revealed
macrocytic anemia which was confirmed on
bone marrow biopsy.
• Vitamin B12 levels in the serum were 7
micrograms (normal range 200 to 600
picograms /ml).
• Gastric endoscopy and biopsy did not reveal
changes of atrophic gastritis.
• Magnetic resonance imaging (MRI) of the spine
showed intramedullary hyperintensity seen on
T2-weighted images in the posterior column of
the cervical spinal cord
 Case 6
• A 29-year-old caucasian male presented to admissions unit complaining of
a 6 week history of dyspnoea on exertion and sweats.
• He had a 7 year history of alcohol excess, drinking 100-200g of alcohol per
day.
• He was a smoker of 10 cigarettes per day, but was otherwise fit and well
with no significant past medical or family history.
• The patient reported no other symptoms and did not take any regular
medication.
• Examination revealed a pulse rate of 124 beats/minute and a blood pressure
of 120 mmHg/48 mmHg.
• He had visibly pale conjunctiva and a lemon yellow tint to the skin and icteric
sclerae, but there were no signs of chronic liver disease.
• On abdominal examination there was palpable splenomegaly but not other
organomegaly or ascites.
• Neurological examination was normal
 Case 6
Haemoglobin (g/dL) 2.8 13.0 – 17.0

Mean cell volume (fL) 112 83 – 105

Platelet count
26 150 – 400
(×109/L)

White cell count

2.6 4.0 – 11.0
(×109/L)

Neutrophils (×109/L) 1.22 2.0 – 7.0

Lymphocyte (×109/L) 1.24 1.0 – 4.0

Monocytes (×109/L) 0.10

0.2 – 1.0
 Folic Acid physiology
• Within the plasma, folate is present, mostly in the
5-methyltetrahydrofolate (5-methyl THFA) form.
• Once inside the cell, 5-methyl THFA may be demethylated to
THFA, the active form participating in folate-dependent
enzymatic reactions. Cobalamin (B-12) is required in this
conversion, and in its absence, folate is "trapped" as 5-
methyl THFA.
• THFA plays a key role in the transfer of 1-carbon units (such
as methyl, methylene, and formyl groups) to the essential
substrates involved in the synthesis of DNA, RNA, and
proteins.
• A healthy individual has about 500-20,000 mcg of folate in
body stores.
• Humans need to absorb approximately 50-100 mcg of folate
per day in order to replenish the daily degradation and loss
through urine and bile. Otherwise, signs and symptoms of
deficiency can manifest after 4 months.
 Folates deficiency causes
• Inadequate ingestion of folate-containing foods (poor
nutrition is prevalent among people with alcoholism)
• Impaired absorption (small bowel resection or
mesenteric vascular insufficiency)
• Impaired metabolism (antimetabolites: methotrexate and
trimethoprim)
• Increased requirement (pregnancy, lactation, rapid
growth period)
• Increased destruction (superoxide, an active metabolite
of ethanol metabolism, is known to inactivate folate)
 Folic Acid deficiency
• Manifestations of folate deficiency would involve impairment
of cell division, accumulation of possibly toxic metabolites
such as homocysteine, and impairment of methylation
reactions involved in the regulation of gene expression, thus
increasing neoplastic risks.
• Folate deficiency can cause anemia. The presentation
typically consists of macrocytosis and hypersegmented
polymorphonuclear leucocytes (PMNs).
• In the scenario of folate deficiency, homocysteine
accumulates. Several recent clinical studies have indicated
that mild-to-moderate hyperhomocystinemia is highly
associated with atherosclerotic vascular disease such as
coronary artery disease (CAD) and stroke.
• Possible pregnancy complications secondary to maternal
folate status may include spontaneous abortion, abruption
placentae, and congenital malformations (eg, neural tube
defect).
 Case 7
• In 2005, this 38-year-old woman lost 125 pounds after
undergoing laparoscopic gastric bypass surgery with
creation of a Roux-en-Y anastomosis.
• She was nonadherent to multivitamin treatment after
undergoing gastric bypass surgery.
• She unexpectedly conceived ~ 1 year after her gastric
bypass.
• Baseline folate and B12 levels were decreased.
• Prenatal ultrasonography revealed an open neural tube
defect and myelomeningocele at approximately L-5 to S-1.
• The patient delivered an otherwise healthy baby girl with a
large myelomeningocele via cesarian section.
 Case 8
• A 59-year-old white male patient with CRF due to diabetic
nephropathy was followed as an out-patient in chronic kidney
disease clinic.
• When he was 47 years old, diabetes mellitus was diagnosed,
and he was treated with oral antidiabetics for 2 years and
thereafter with insulin.
• At 55, a severe polyneuropathy and distal occlusive arterial
disease with foot gangrene occurred that required the
amputation of the left foot.
• At the age of 59, during a visit, the nephrologists noticed
speech disturbances, mostly stuttering.
• He had a normocytic, normochromic anaemia (Hb = 9.2
mg/dl, MCV = 95 fl), severe CRF (creatinine = 5.4 mg/dl),
hyperkalaemia (6 mEq/l) and a normal serum Ca (4.7 mEq/l).
 Case 9
• A 12-year-old male presents with high fever, marked pallor, and
epistaxis;
• he has a history of recurrent URIs and high-grade fever that
have been treated with parenteral antibiotics
(Chloramphenicol).
• He has also shown marked weakness over the past 3 months.
• He lives in the vicinity of an industrial unit that handles
petroleum distillates such as benzene.
• PE: fever; marked pallor of skin and conjunctiva; oral and nasal
mucosal petechiae; purpuric patches visible on skin; no
significant lymphadenopathy; no hepatosplenomegaly.
• Labs: anemia, neutropenia, and thrombocytopenia
(PANCYTOPENIA); anemia with low reticulocyte count; normal
RBC morphology.
• Normal serum bilirubin;
• Micro Pathology of bone marrow: Hypocellular bone marrow
with empty spaces populated by fat cells, fibrous stroma and
scattered lymphocytes; marked decrease in all cell lines.
 Aplastic anemia
• Aplastic anemia is a syndrome of bone
marrow failure characterized by peripheral
pancytopenia and marrow hypoplasia

• Basis for marrow failure includes primary

defects in or damage to the stem cell or
the marrow microenvironment.
 Aplastic anemia causes
Congenital causes of aplastic anemia (20%):
• Fanconi anemia
• Dyskeratosis congenita

Acquired causes of aplastic anemia (80%):

• Idiopathic factors
• Infectious causes, such as hepatitis viruses, EBV, HIV
• Toxic exposure to radiation and chemicals, such as benzene
• Drugs, such as chloramphenicol
 Fanconi Anemia
• Fanconi anemia (FA) is the most
frequently reported of the rare inherited
bone marrow failure syndromes

• In 1927, Guido Fanconi first reported 3

brothers with pancytopenia and physical
abnormalities.
 Fanconi anemia manifestation
• About 75% of patients with Fanconi anemia
have birth defects:
– altered skin pigmentation and/or café au lait spots (>50%),
– short stature (50%),
– thumb or thumb and radial abnormalities (40%),
– abnormal male gonads (30%),
– microcephaly (25%),
– eye anomalies (20%),
– developmental delay (10%),
– abnormal ears or hearing (10%).
• Pancytopenia
 FA pathophysiology
• Fanconi anemia is an autosomal recessive disease in more
than 99% of patients (FANCB is X-linked recessive); each
patient with Fanconi anemia is homozygous or doubly
heterozygous for mutations in one of the 13 genes known to
be responsible for FA.

• The Fanconi anemia proteins A, B, C, E, F, G, L and

M appear to form a nuclear complex, which leads to
ubiquitination of the I and D2 proteins; the latter is involved
in DNA damage response mechanisms in cooperation with
FANCD1, FANCJ, and FANCN, as well as BRCA1, RAD51,
Mre11, and other proteins.

• The specific role of mutations in the Fanconi anemia genes

in the pathogenesis of birth defects, bone marrow failure, or
oncogenesis is not yet clear.
 FA pathophysiology
• Fanconi anemia cells may be susceptible
to damage by oxygen free radicals.
• Fanconi anemia cells have a defect in cell
cycle regulation.
• The hematopoietic stem cell is defective in
Fanconi anemia.
• A defect in the DNA-damage response
pathway is present in Fanconi anemia.
 Case 10
• 25-year-old woman presents with high-grade fever,
menorrhagia, and marked weakness.
• Over the past several weeks, she has also had recurrent
infections.
• PE: Marked pallor; multiple purpuric patches over skin;
hepatosplenomegaly; normal fundoscopic and neurologic
exam.
• Labs: normocytic, normochromic anemia; thrombocytopenia;
leukocytosis composed mainly of myeloblasts and
promyelocytes (nonmaturing, early blast cell ); neutropenia.
– Hb 3,3g/dl
– Plt 9000/mcl
– WBC 20,000/mcl
• Micro Pathology of bone marrow: Myeloblasts with
myelomonocytic differentiation replace normal marrow;
peroxidase-positive stains on bone marrow and gingival
biopsy.
 Myelophthisic Anemia
• Myelophthisis is a form of bone marrow failure that results
from the destruction of bone marrow precursor cells and their
stroma, caused by nonhematopoietic cells invading bone
marrow.

• Can result in varying degrees of cytopenia, including anemia,

thrombocytopenia, neutropenia, and pancytopenia.

• The most common causes of infiltrative myelopathy are

metastatic carcinomas (eg, lung, breast, and prostate
cancer), lymphoproliferative malignancies (eg, lymphomas),
disseminated granulomatous diseases (eg, tuberculosis)
 Case 11
• A 9-month-old infant is brought to the
pediatrician because of jaundice, Iethargy,
and easy fatigability.
• The parents of that child are immigrants of
northern European origin.
• PE: pallor; mild jaundice; palpable
splenomegaly.
• microcytic anemia; elevated indirect bilirubin;
increased reticulocytes; abnormal RBC
osmotic lysis test.
Case 11
 Congenital spherocytosis
• Hereditary spherocytosis (HS) is a familial
hemolytic disorder with marked
heterogeneity of clinical features, ranging
from an asymptomatic condition to
fulminant hemolytic anemia.
• The morphologic hallmark of HS is the
microspherocyte, which is caused by loss
of membrane surface area, and an
abnormal osmotic fragility in vitro.
 Congenital spherocytosis
• HS erythrocytes are caused by membrane protein defects
resulting in cytoskeleton instability. Spectrin deficiency leads
to loss of erythrocyte surface area, which produces spherical
RBCs.

• Spherocytic RBCs are culled rapidly from the circulation by

the spleen. Patients with HS develop splenomegaly.

• Mutations of alpha-spectrin are associated with recessive

forms of HS, whereas mutations of beta-spectrin occur in
families with autosomal dominant forms of HS
RED CELL CYTOSKELETON
 Hereditary Elliptocytosis
• Hereditary elliptocytosis (HE) and its variants
are congenital hemolytic disorders in which erythrocytes
are either elongated into a cigar or oval shape or are
poikilocytic and bizarrely shaped.
• Mutations that disrupt the formation of spectrin tetramers
result in hereditary elliptocytosis.
• Its transmission has usually been described
as autosomal dominant.
• The result of these mutations is a mechanically unstable
membrane less tolerant of shear stress that is
susceptible to permanent deformation.
Elliptocytes/ovalocytes
 HEMOLYTIC ANEMIA
• Anemia of increased destruction
– Normochromic, normocytic anemia
– Shortened RBC survival
– Reticulocytosis - Response to increased RBC
destruction
– Increased indirect bilirubin
– Increased LDH
 HEMOLYTIC ANEMIA
Causes
• INTRACORPUSCULAR HEMOLYSIS
– Membrane Abnormalities
– Metabolic Abnormalities
– Hemoglobinopathies
• EXTRACORPUSCULAR HEMOLYSIS
– Nonimmune
– Immune
 Case 12
• A 10-year-old black child presents with a chronic
nonhealing ulcer on his lower Ieg.
• He ha had recurrent episodes of abdominal and chest
pain along with diminution of vision.
• His maternal cousin suffer from a blood disorder.
• PE: pallor; mild icterus; fundoscopy revealed hypoxic
spots with neovascularization
• Labs: decreased hematocrit; megaloblastic anemia,
serum bilirubin moderately elevated; abdomen: small
calcified spleen
Case 12
 Sickle cell anemia
• Sickle cell anemia is one of the most
prevalent genetic disorders world-wide
• Autosomal recesive type of inheritance
• Results from a variant of the Beta Globin
gene on chromosome 11
– a glutamic acid at the 6th position is replaced by
a valine resulting in formation of Hemoglobin S
• Sickle cell disease is a form of hemolytic
anemia with red cell survival of around 10-20
days.
Hemoglobin A
Normal hemoglobin has
four subunits that each
contain an oxygen binding
site.
Hemoglobin S
A single mutation in
hemoglobin results in a
binding of one protein
to another.
•HbS has the physical properties of forming
polymers under deoxy conditions.
•Under deoxy conditions, Hb S undergoes
marked decrease in solubility, increased
viscosity, and polymer formation.
•When red blood cells (RBCs) containing
homozygous Hb S are exposed to deoxy
conditions, the sickling process begins.
•After recurrent episodes of sickling,
membrane damage occurs and the cells
are no longer capable of resuming the
biconcave shape upon reoxygenation.
Thus, they become irreversibly sickled cells
(ISCs). From 5-50% of RBCs permanently
remain in the sickled shape.
Sickle cell with normal RBC
 SCD – ishaemic complications
• Growing evidence indicates that sickle cell disease is a
state of inflammation characterized by vascular
endothelium activation and increased blood cell–
endothelium interactions.
• Abnormal interaction of sickle red cells with vascular
endothelium is considered a key contributor to the
initiation of vaso-occlusion in this disease.
• Rigid cells progressively obstruct and damage the
spleen, which leads to functional asplenia. This, along
with other abnormalities, results in extreme susceptibility
to infection.
 Case 13
• An 11-month-old male presents with marked pallor, failure to
thrive, and delayed developmental motor milestones.
• The child's parents are Italian immigrants.
• Marked pallor; mild icterus; frontal bossing and maxillary
hypertrophy; splenomegaly.
• severe microcytic, hypochromic anemia with
anisopoikilocytosis; decreased reticulocytosis; mildly increased
unconjugated bilirubin
• HbA absent; HbF 95%.
• Gross Pathology: Expansion of hematopoietic bone marrow,
causing thinning of cortical bone or new bone formation.
• Micro Pathology: Red marrow increased; yellow marrow
decreased; marked erythroid hyperplasia in marrow (ineffective
erythropoiesis).
 Thalassemia
• Thalassemia = Mediterranean anemia - in
1925, Thomas Cooley, a Detroit
pediatrician, described a severe type of
anemia in children of Italian origin.

• The thalassemias are inherited disorders

of hemoglobin (Hb) synthesis.
• To date, more than 1000 inherited
mutations that affect either the structure or
synthesis of the α- and β-globin chains are
known.
 Thalassemia pathophysiology
• The thalassemias are inherited disorders of Hb synthesis that
result from an alteration in the rate of globin chain production.
• A decrease in the rate of production of a certain globin chain or
chains (α, β, γ, δ) impedes Hb synthesis and creates an
imbalance with the other, normally produced globin chains.
• 2 types of chains (α and non-α) pair with each other at a ratio
close to 1:1 to form normal Hb.
• The consequences of impaired production of globin chains
ultimately result in the deposition of less Hb into each RBC,
leading to hypochromasia.
• An excess of the normally produced type is present and
accumulates in the cell as an unstable product, leading to the
destruction of the cell.
 Thalassemia types
• The type of thalassemia usually carries the
name of the underproduced chain or chains.
– β thalassemia – thalassemia major – severe type
– α thalassemia - thalassemia minor – mild type
 Thalassemia pathophysiology
• In the most common type of β thalassemia trait, the level of
Hb A2 (δ2/α2) is usually elevated.
• This is due to the increased use of δ chains by the excessive
free α chains, which results from a lack of adequate β chains
with which to pair.
• The remaining α chains precipitate in the cells, reacting with
cell membranes, intervening with normal cell division, and
acting as foreign bodies, leading to destruction of RBCs.
• This leads to excessive intramedullary destruction of the RBC
precursors.
• In addition, the surviving cells that arrive in the peripheral
blood with intracellular inclusion bodies (excess chains) are
subject to hemolysis.
Thalassemia pathophysiology
• Hypoxia, together with the profound
anemia, stimulates the production of
erythropoietin. As a result, severe
expansion of the ineffective erythroid mass
leads to severe bone expansion and
deformities.
• The large numbers of abnormal RBCs
processed by the spleen results in
massive splenomegaly, leading to
manifestations of hypersplenism.
Thalassemia – blood analysis
• Mild anemia with marked
hypochromia and
microcytosis (without the
anisocytosis usually
encountered in iron
deficiency).
• abnormal Hb
electrophoresis (elevated
levels of Hb A2, Hb F, or
both).
• Target cells.
Bones abnormalities in
Thalassemia
 Case 14
• 4 year old black boy from Venezuela was admitted to the
pediatric clinic in Lodz with headache and vomiting.
• He was acutely ill and was passing dark urine.
• Sixty hours earlier he had eaten fresh fava beans
• PE: tachycardia, pallor, mild jaundice, no heapatomegaly
or splenomegaly.
• Labs: Hb – 5,5g/dl; retikulocytes 7%; single spherocytes;
increased indirect bilirubin
• Urinalysis: massive hemoglobinuria
Vicia faba, also known
as the broad bean
Heinz bodies, composed of denatured hemoglobin
 Glucose-6-Phosphate
Dehydrogenase Deficiency
Favism
• Glucose-6-phosphatase dehydrogenase
(G6PD) deficiency is the most common
disease-producing enzymopathy in
humans.
• Inherited as an X-linked disorder.
• Affects 400 million people worldwide.
Glucose 6-Phosphate Dehydrogenase
• Regenerates NADPH, allowing regeneration of
glutathione and normal function of glutathione
peroxydase
• Protects against oxidative stress
• Lack of G6PD leads to Coombs’ Negative
hemolysis during oxidative stress
– Infection
– Medications (oxidative drugs – ex. Sulfonamides or
antimalaric drugs)
– Fava beans
• Oxidative stress leads to Heinz body formation,
intratravascular hemolysis
 G6PD DEFICIENCY
Function of G6PD

Infections
Drugs
2 H2O H2O2 Hgb

Sulf-Hgb
GSSG 2 GSH

NADPH NADP Heinz bodies

6-PG G6PD
G6P
Hemolysis
 Case 15
• A 19-year-old male comes to see the nurse at the
college health department complaining of abdominal and
Iumbar pain, which characteristically occurs when he
takes his multivitamin pills two times a week
• He has also noticed dark brown urine the morning after
he has the pain.
• He has just left his parents to go to college and is excited
about his newfound freedom; he likes to drink excessive
amounts of beer.
 Case 15
• PE: Marked pallor; the rest of examination
unremarkable
• Labs: normocytic, normochromic hemolytic
anemia with reticulocitosis .
• Hemoglobinemia and hemoglobinuria;
acidified serum test positive (HAM'S TEST).
Paroxysmal Nocturnal Hemoglobinuria

• PNH is a descriptive term for the clinical manifestation of

red blood cell breakdown with release of hemoglobin into
the urine that is manifested most prominently by dark-
colored urine in the morning.
• The term "nocturnal" refers to the belief that hemolysis is
triggered by acidosis during sleep and activates
complement to hemolyze an unprotected and abnormal
RBC membrane.
• Hemolysis has been shown to occur throughout the day and
is not actually paroxysmal, but the urine concentrated
overnight produces the dramatic change in color.
 PNH manifestation
• The clinical syndrome can present in 3 types of
symptoms including:
– an acquired hemolytic anemia due to the abnormal
susceptibility of the RBC membrane to the
hemolytic activity of complement;
– thromboses in large vessels, such as hepatic,
abdominal, cerebral, and subdermal veins;
– a deficiency in hematopoiesis that may be mild or
severe, such as pancytopenia
 PNH pathophysiology
• X-linked dominant type of inheritance
• Genetic mutation leading to the inability to
synthesize the glycosyl-phosphatidylinositol
(GPI) anchor that binds proteins to cell
membranes in hematopoietic stem cells
• Loss of membrane proteins which inhibit the
function of complement and protect normal
cells
 PNH thrombotic complications
• Breakdown of RBC membranes by complement leads to
the release of hemoglobin into the circulation.
• After saturating the haptoglobin, free forms of
hemoglobin circulates and binds irreversibly with nitric
oxide (NO) and depletes NO levels in peripheral blood.
• Because NO is a smooth muscle tone regulator,
depletion of NO levels leads to smooth muscle
contraction with consequent vasoconstriction,
constriction of the gut, and pulmonary hypertension.
• Platelets can be abnormaly activated by complement
and may exibit increased agregation and coagulation
 Case 16
• A 66-year-old white man recently diagnosed with chronic
lymphocytic leukemia comes into the emergency room
complaining of fatigue and tachycardia.
• He also states that his urine has been progressively
turning dark and red over the course of the day.
• PE : tachycardia; dyspnea; pallor of skin and mucous
membranes; slight jaundice; splenomegaly.
• Labs: severe anemia; positive Coombs' test;
reticulocytosis; hemoglobinuria.
• Increased serum indirect bilirubin.
IMMUNE HEMOLYTIC ANEMIA
General Principles
• All require antigen-antibody reactions
• Types of reactions dependent on:
– Class of Antibody
– Number & Spacing of antigenic sites on cell
– Availability of complement
– Environmental Temperature
– Functional status of reticuloendothelial system
• Manifestations
– Intravascular hemolysis
– Extravascular hemolysis
 IMMUNE HEMOLYTIC ANEMIA
• Drug-Related Hemolysis
• Alloimmune Hemolysis
– Hemolytic Transfusion Reaction
– Hemolytic Disease of the Newborn
• Autoimmune Hemolysis
– Warm autoimmune hemolysis
– Cold autoimmune hemolysis
• Often associated with either lymphoproliferative
disease or collagen vascular disease
 IMMUNE HEMOLYTIC ANEMIA
Three main types exist:
• warm antibody (80% to 90%; associated with leukemia,
lymphoma, SLE, and viral infections);
– Most warm autoantibodies are immunoglobulin (Ig) G and
can be detected with the direct Coombs test
• cold reacting antibody (10%; associated with EBV;
mycoplasma infections and lymphoma)
– Most commonly IgM mediated
– Antibodies bind best at 30º or lower
– Fix entire complement cascade
– Leads to formation of membrane attack complex, which
leads to RBC lysis in vasculature
– Typically only complement found on cells
• drug-induced (methyldopa, quinidine, penicillin).
IMMUNE HEMOLYTIC ANEMIA
Coombs Test - Direct

• Looks for immunoglobulin and/or complement of

surface of red blood cell (normally neither found
on RBC surface)
• Coombs reagent - combination of anti-human
immunoglobulin and anti-human complement
• Mixed with patient’s red cells; if immunoglobulin
or complement are on surface, Coombs reagent
will link cells together and cause agglutination of
RBCs
 IMMUNE HEMOLYTIC ANEMIA
Coombs Test - Indirect

• Looks for anti-red blood cell antibodies in the

patient’s serum, using a panel of red cells
with known surface antigens
• Combine patient’s serum with cells from a
panel of RBC’s with known antigens
• Add Coombs’ reagent to this mixture
• If anti-RBC antigens are in serum,
agglutination occurs
 IMMUNE HEMOLYTIC ANEMIA
• Antibodies combine with RBC, and either
― Activate complement cascade, and/or
― Opsonize RBC for immune system
• if all of complement cascade is fixed to
red cell, intravascular cell lysis occurs
• if complement is only partially fixed,
macrophages recognize Fc receptor of Ig
or C3b of complement and phagocytize
RBC, causing extravascular RBC
destruction
 Case 17
• 18-year-old hospitalized male complains of fever,
nausea, vomiting, and chest pain following a blood
transfusion.
• He was involved in a motorcycle accident and was
rushed to the emergency room, where he received five
units of blood before being taken to the OR for repair of
a ruptured spleen and liver.
• PE : fever; no hepatosplenomegaly or lymphadenopathy;
surgical laparotomy wound unremarkable.
• Labs: Positive Coombs' test; elevated indirect bilirubin;
cola-colored urine (due to hemoglobinuria).
 ALLOIMUNE HEMOLYSIS
Hemolytic Transfusion Reaction
• Caused by recognition of foreign antigens on
transfused blood cells
• Several types
– Immediate Intravascular Hemolysis (Minutes) - Due to
preformed antibodies; life-threatening
– Slow extravascular hemolysis (Days) - Usually due to
repeat exposure to a foreign antigen to which there was
a previous exposure; usually only mild symptoms
– Delayed sensitization - (Weeks) - Usually due to 1st
exposure to foreign antigen; asymptomatic
 ALLOIMMUNE HEMOLYSIS
Hemolytic Disease of the Newborn
• Due to incompatibility between mother negative for an
antigen and fetus positive for that antigen.
• Rh incompatibility, ABO incompatibility most common
causes
• Usually occurs with 2nd or later pregnancies
• Requires maternal IgG antibodies vs. RBC antigens in fetus
• Can cause severe anemia in fetus, with erythroblastosis and
heart failure
• Hyperbilirubinemia can lead to severe brain damage
(kernicterus) if not promptly treated
• HDN due to Rh incompatibility can be almost totally
prevented by administration of anti-Rh D to Rh negative
mothers after each pregnancy
 Case 18
• A 33-year-old woman gave birth at term to a
male baby (weight 3.7 kg, length 51 cm) by the
vaginal route.
• Prenatal and natal histories were normal.
• Postpartum examination revealed a giant
hemangioma extending to the right arm
centralizing at the elbow.
• Its surface was not intact; there was an ulcer
wound that sometimes bled and there was a
small, infected lesion on the surface of the giant
hemangioma
 Case 18
• Laboratory data revealed findings of severe
consumptive coagulopathy. Upon admission, the
hemoglobin level was low (9.6 g/dl); erythrocyte
(2.9·106/mm3) and severe thrombocytopenia
(14000/mm3) were recorded, as well as a
coagulopathy with abnormal activated partial
thromboplastine time (aPTT: 61 s, n.v. 25–45 s.),
and very low fibrinogen (30 mg/dl n.v. 50–250
mg/dl). Fibrin degradation products like d-dimer
were increased (9.78 µg/ml n.v. 1–2 mg/dl).
• The peripheral smear was remarkable for a
decreased platelet number and some red blood cell
fragments.
Schistiocytes in peripheral blood
 Microangiopathic Hemolysis
• Thrombotic microangiopathies are characterized
by the development of hyaline thrombi in small
vessels resulting in thrombocytopenia,
microangiopathic hemolysis, and organ
dysfunction (Thrombotic Thrombocytopenic
Purpura)
• Kasabach-Merritt syndrome is characterized by
thrombocytopenia, microangiopathic hemolytic
anemia, consumption coagulopathy and
progressively extending vascular lesions.
 Anemia of chronic disease
(ACD) - definition
• ACD is a common type of anemia that
occurs in patients with infectious,
inflammatory, or neoplastic diseases that
persist for more than 1 or 2 months.
• It does not include anemias caused by
marrow replacement, blood loss, hemolysis,
renal insufficiency, hepatic disease, or
endocrinopathy, even when these disorders
are chronic.
 Anemia of chronic disease (ACD) -
epidemiology
The ACD is extremely common
• ACD is more common that any anemia syndrome
other than blood loss with consequent iron deficiency
• ACD is the most common cause of anemia in
hospitalized patients
• After patients with bleeding, hemolysis, or known
hematologic malignancy were excluded, 52% of
anemic patients met laboratory criteria for the anemia
of chronic disorders
• ACD is observed in 27% of outpatients with
rheumatoid arthritis and in 58% of new admissions to
hospital rheumatology units
 Anemia of chronic disease (ACD) -
laboratory features
• The anemia is usually mild or moderate ( Hb 7-11g/dl)
- lower values are observed in 20-30% of patients

• The anemia is most often normochromic and

normocytic (MCHC and MCV are normal)

• Erythrocyte sedimentation rate (ESR) - usually rapid

• Retikulocytes - most often normal or slightly decreased

number.
 Anemia of chronic disease (ACD) -
laboratory features
Iron metabolism
• Serum Iron - decreased (it is necessary for the diagnosis
of ACD)
• TIBC - reduced or low-normal (N)
• Transferrin saturation(TS) - moderately decreased
(higher than in iron-deficiency anemia), usually > 10%
• Serum Ferritin –increased or normal
• Sideroblasts in the bone marrow-reduced (5-20%)
 Anemia of chronic disease (ACD) -
pathogenesis
• Shortened red cell life span, moderately 20-30%
(from 120 to 60-90 days)
• Relative bone marrow (erythropoiesis) failure
- Cytokines released from inflammatory cells (TNF-a,
IL-1, IFN-g) affects erythropoiesis by inhibiting the
growth of erythroid progenitors
- Serum erythropoietin levels in patiens with ACD are
normal when compared to healthy subjects but much
lower than levels in non-ACD anemic patients
Anemia of chronic disease (ACD) -
pathogenesis

ABNORMAL IRON METABOLISM

• Activation of the reticuloendothelial system with
increased iron retention and storage within it
• impaired release of iron from macrophages to
circulating transferrin (impaired reutilization of iron)
• Reduced concentration of transferrin
(decreased production, increase sequestration in the
spleen and in the foci of inflammation, increase loss )
 Adventages of ACD
• Withdrawal of iron by increased storage of the metal
within the reticuloendothelial system acts to limit the
availability of iron to microorganisms or tumor cells
and thereby inhibit their growth and proliferation

• Decreased hemoglobin reduces the oxygen transport

capacity of the blood and decreases the overall
oxygen supply, which may primarily affect rapid
proliferating (malignant) tissues and micro-organism

• Retention and storage of iron in reticuloendothelial

system directly and indirectly via cytokines strongly
affects cell mediated immune function