Hematological diseases

● Anemia is a state of decreased red cell mass of blood leading to the decreased
oxygen-carrying capacity of the body. Red cell indices are useful in classifying anemia.
Anemias are classified etiologically and morphologically.
● Anemia is defined as the state in which the red cell mass of blood is decreased
below the normal level for the age and sex of the patient. As a result of this, the
oxygen-carrying capacity of blood is decreased. It is characterized by a decrease in
hemoglobin, packed red cell volume (PCV), and red blood cell (RBC) count.
● It is a common condition that can be caused by inadequate RBC production,
excessive RBC destruction, or blood loss. The most common cause is iron deficiency.
Clinical features, if present, are mostly nonspecific and may include fatigue, dyspnea(a
sensation of breathing discomfort or shortness of breath), conjunctival
pallor(conjunctival = a highly vascularized mucous membrane that lines the inside of
eyelids and sclera, pallor = a morphological descriptor for paleness of the skin and
mucous membrane), and tachycardia (increased heart rate).
● Once anemia has been established, the mean corpuscular volume (MCV) (the
average volume of red blood cells which is around 80-100ml) should be checked to
distinguish between microcytic(a descriptor for cells of abnormally small size),
normocytic ( a descriptor for erythrocytes of normal sizes), and macrocytic anemia (a
type of anemia is characterized by larger than normal red blood cells) and to
determine the next diagnostic steps. A reticulocyte count can also be used to evaluate
the bone marrow response. Treatment depends on the form of anemia and underlying
condition. Acute and/or severe cases of anemia may require transfusion of packed
red blood cells.
1. Iron deficiency
a. Definition
Iron deficiency occurs when the body's iron stores are insufficient for the normal
formation of Hb, iron-containing enzymes, and other functional iron compounds such
as myoglobin and those of the cytochrome system. Iron deficiency can be classified
according to its severity: normal stores; negative iron balance; iron store depletion (low
serum ferritin); decreased serum iron [low serum iron, increased total iron-binding
capacity (TIBC)]; and anemia (reduced Hb with microcytic, hypochromic erythrocytes).3
Erythrocytes of patients with mild, early-stage iron deficiency often appear to be
normal in color and size (i.e., normochromic, normocytic).
Body iron usually is kept constant by a delicate balance between the amount lost and
absorbed. There is no physiologic mechanism for excreting iron in humans.
Consequently, there is only a limited ability to compensate for excessive loss or
absorption of iron. Iron balance is a conservative system, and in the normal adult,
even if iron intake is negligible, it takes at least 2 to 3 years to develop iron deficiency.
b. Role of iron
Storage iron (0.3–1.5 g), in the form of ferritin and hemosiderin, is located mainly in the
parenchymal cells of the liver, the reticuloendothelial cells of the spleen, and bone
marrow, and it replenishes functional iron. Iron stores account for one-third of body
iron in healthy men. Iron stores are more variable and are generally lower in children
and women of childbearing potential. Low iron stores are an early sign of iron
deficiency and may help differentiate between iron deficiency anemia and other
causes of anemia.
About 70 percent of your body's iron is found in the red blood cells of your blood
called hemoglobin and in muscle cells called myoglobin. Hemoglobin is essential for
transferring oxygen in your blood from the lungs to the tissues. Myoglobin, in muscle
cells, accepts, stores, transports, and releases oxygen.
About 6 percent of body iron is a component of certain proteins, essential for
respiration and energy metabolism, and as a component of enzymes involved in the
synthesis of collagen and some neurotransmitters. Iron also is needed for proper
immune function.
About 25 percent of the iron in the body is stored as ferritin, found in cells, and
circulates in the blood.
c. Sources of iron
● Meat: beef, pork, or lamb, especially organ meats such as liver
● Poultry: chicken, turkey, and duck, especially liver and dark meat
● Fish, especially shellfish, sardines, and anchovies
● Leafy green members of the cabbage family including broccoli, kale,
turnip greens, and collard greens
● Legumes, including lima beans, peas, pinto beans, and black-eyed peas
● Iron-enriched pasta, grains, rice, and cereals
● The amount of iron needed to treat patients with iron deficiency is higher than
the amount found in most daily multivitamin supplements.
● Intravenous iron(IV) = IV iron comes in different preparations: Iron dextran, Iron
sucrose, and Ferric gluconate
d. Absorption of iron
Iron absorption is regulated by iron needs and body stores. When iron stores are low
or depleted, a higher proportion of available iron is absorbed. Absorption decreases
when the stores are replete. The serum ferritin concentration, which reflects body iron
stores, is inversely related to iron absorption. However, this feedback process can be
overwhelmed when large amounts of iron are presented for absorption (e.g., in iron
overdose or toxicity cases). In some clinical states, such as primary hemochromatosis,
thalassemia, and sideroblastic anemia, iron absorption remains normal and even
elevated despite increased iron stores.
The iron content of food and its bioavailability determine if the diet can meet
physiological needs. Dietary iron is present in two major pools: heme iron and
non-heme iron. Heme iron, found only in meats, is two to three times more absorbable
than nonheme iron, found in plant-based and iron-fortified foods. Ingested heme
compounds and organic nonheme iron complexes are broken down in the acid
environment of the stomach to ferric ions (Fe3+) and heme molecules, respectively. The
stomach's acidity promotes the reduction of iron from the ferric state to the ferrous
state (Fe2+), which is better absorbed. Patients with achlorhydria secondary to age or
gastrectomy tend to absorb nonheme iron poorly. Iron is absorbed primarily in the
upper duodenum. The iron-absorptive capacity is limited by the rate at which iron is
transferred from the intestinal lumen to the plasma. The reduced (ferrous) iron binds
to specific sites on the lumen and is actively carried across the intestinal membrane.
Iron absorbed by these cells is incorporated into an iron carrier pool, most of which is
deposited as ferritin or used by the mitochondria for enzyme synthesis. A small
amount of iron is lost through the normal sloughing of the mucosal cells in the
gastrointestinal tract. A smaller proportion of the iron from the carrier pool is
transferred to the plasma, where the ferric form binds tightly to transferrin.
A number of factors can inhibit or promote iron absorption. Foods that can reduce
iron absorption by forming less soluble complexes include coffee, tea, milk, and milk
products, eggs, whole-grain bread and cereals, and any food containing
bicarbonates, carbonates, oxalates, or phosphates. Commercial processing or
enhancers can improve absorption from food in some cases. Enhancers of nonheme
iron absorption are food acids such as citric, lactic, or ascorbic acids, and meats.
Ascorbic acid, the most powerful promoter, has a dose-related effect on nonheme iron
absorption. In its presence, ferric iron is converted to the ferrous state, maintaining
iron solubility in the alkaline environment of the duodenum and upper jejunum.
Ascorbic acid also forms an alkaline-stable chelate with ferric chloride in the stomach.
Meat, itself a rich source of iron, also promotes the absorption of nonheme iron.
Approximately 1 g of meat enhances nonheme iron absorption to about the same
extent as 1 mg of ascorbic acid. Citric acid, a common food additive and a less
powerful promoter of iron absorption has an additive effect on ascorbic acid
e. Metabolism of iron
❖ Iron absorption
Iron is absorbed in the duodenum and upper jejunum. The absorption of iron is
determined by the type of iron molecule and by what other substances are
ingested. Iron absorption is best when food contains heme iron (meat). Dietary
nonheme iron is usually in the ferric state and must be reduced to the ferrous
state and released from food binders by gastric secretions. Nonheme iron
absorption is reduced by other food items (eg, vegetable fiber phytates and
polyphenols; tea tannates, including phosphoproteins; bran) and certain
antibiotics (eg, tetracycline). Ascorbic acid is the only common food element
known to increase nonheme iron absorption.
❖ Iron transport and usage
Iron is absorbed in the duodenum and upper jejunum. The absorption of iron is
determined by the type of iron molecule and by what other substances are
ingested. Iron absorption is best when food contains heme iron (meat). Dietary
nonheme iron is usually in the ferric state and must be reduced to the ferrous
state and released from food binders by gastric secretions. Nonheme iron
absorption is reduced by other food items (eg, vegetable fiber phytates and
polyphenols; tea tannates, including phosphoproteins; bran) and certain
antibiotics (eg, tetracycline). Ascorbic acid is the only common food element
known to increase nonheme iron absorption.

❖ Iron storage and recycling

 Iron not used for erythropoiesis is transferred by transferrin to the storage
pool; iron is stored in 2 forms:
● Ferritin
● Hemosiderin
The most important is ferritin (a heterogeneous group of proteins
surrounding an iron core), which is a soluble and active storage fraction located
in the liver (in hepatocytes), bone marrow, and spleen (in macrophages); in red
blood cells (RBCs); and in serum. Iron stored in ferritin is readily available for
anybody’s requirement. Circulating (serum) ferritin level parallels the size of the
body stores (1 ng/mL = 8 mg of iron in the storage pool).

The 2nd storage pool of iron is in hemosiderin, which is relatively insoluble and
is stored primarily in the liver (in Kupffer cells) and in the bone marrow (in
macrophages).

Because iron absorption is so limited, the body recycles and conserves iron.
Transferrin grasps and recycles available iron from aging RBCs undergoing
phagocytosis by mononuclear phagocytes. This mechanism provides about 97%
of the daily iron needed (about 25 mg of iron).

f. Symptoms
● Dyspnoea
● Fatigue
● Headache
● Dizziness
● Syncope
● Confusion
● Palpitations
● Angina
● Tiredness, weakness, or fainting
● Breathlessness
g. Inadequate dietary intake
● The deficient iron content of the food: This is common in infants who are kept
too long exclusively on a milk diet and in native populations living on marginal
and poor diets. Iron deficiency may occur in older people due to their limited food
intake like meat due to dental problems and poverty.
● Deficient absorption of iron: Deficient absorption usually follow,
○ Poor dietary practice like, less consumption of diets rich in vitamin C
which enhance iron absorption,
○ Drinking coffee and tea immediately after meal inhibits iron absorption,
○ Gastrointestinal tract operation. It may occur also in chronic
malabsorption states or diseases and consumption of antacids, fibrous diet,
and heavy metals like calcium, zinc, magnesium.
● Deficient transport: A decrease in transferring (iron-binding protein) is
associated with a number of inflammatory conditions particularly rheumatoid
 arthritis. This may result in a decrease body iron content and finally production of
less pigmented (hypochromic) red blood cells.
● Abnormal loss of iron: It is commonly caused by loss of circulating red cells
through a hemorrhage, excessively heavy menstruation, or parasites like
hookworm and schistosomiasis.
● Increased physiologic requirements: This occurs primarily in children during
active growth and in pregnant women. When the infant is put on a prolonged
exclusive milk diet, the need for iron is not met. Pre-term infants require more iron.
Pregnancy and lactation also places heavy demands on the iron stores of the
mother
h. Pathogenesis
● Iron store depletion: The first stage in the development of iron deficiency
anemia is depletion of body iron stores. At this stage, the patient does not have
typical clinical and laboratory findings of iron deficiency anemia.
● Iron deficient erythropoiesis (RBC production). This stage is marked by
limitations in RBC production. Still in this stage, there is no typical laboratory
finding of iron deficiency anemia which is a microcytic-hypochromic picture in
RBC.
● Iron deficiency anemia: This stage indicates a prolonged negative iron
balance (the requirement and/or the loss of iron exceeds the intake) and this
eventually results in the production of poorly hemoglobinized cells (hypochromic -
microcytic red blood cell morphology).
i. Lab diagnosis
I. diagnosis of anemia
Anemia can be identified using the following methods:
● History: Detect clinical symptoms of anemia, dietary history.
● Physical Examination: Examine mucous membrane (mouth, conjunctiva),
palm, and fingernails
● Laboratory Examination: Measure hemoglobin or hematocrit.
II. diagnosis of causes
The possible causes of anemia are traced with the following steps.
● History: age, sex, pregnancy, diet, clinical symptoms, etc.
● Physical examination: e.g. Signs of infection such as malaria.
● Laboratory examination:
○ Blood film for hemiparasites like malaria
○ Peripheral blood morphology
○ Stool examination for hookworm and schistosomiasis
○ Urinalysis
○ Pregnancy test

j. Iron profile
 Iron Profile is a panel of blood tests that check the level of iron in the blood. Iron
Profile shows how much of the iron is stored in the tissues. Iron Profile includes
Serum Iron (Fe), Total Iron Binding Capacity, and % Transferrin Saturation.

k. Clinical features
● Tiredness, weakness, or fainting
● Fatigue
● Breathlessness (shortness of breath)
● Exercise intolerance
● Head ache
● Tinnitus (ringing in the ear)
● Blurred vision
● Nausea
● Poor appetite
● Palpitation (uncomfortable awareness of one’s heartbeat).
● Excessive desire to eat unusual substances (pica) such as clay or ice.
● Paleness (skin and mucus membranes)
● Edema in chronic and sever cases
● Irritability
● Poor growth and development in children
2. Megaloblastic anemia
The molecular basis for megaloblastic is a failure in the synthesis and assembly of
DNA. The most common causes of megaloblastic are cobalamin and folate
deficiencies. Cobalamin metabolism and folate metabolism are intricately related,
and abnormalities in these pathways are believed to lead to the attenuated
production of DNA.

Methotrexate-induced megaloblastic has been ascribed to a deficiency in

deoxythymidine triphosphate (dTTP) that is consumed by the methyl folate trap.
Evidence exists that megaloblastic is caused by interference of folate metabolism by
the inhibition of methionine synthesis. However, because of dietary folate deficiency,
the size of the dTTP pool is normal or increased in persons with megaloblastic.

Impairment in the deoxyuridine monophosphate (dUMP) ¡ú deoxythymidine

monophosphate (dTMP) pathway may be responsible for nutritional megaloblastic.
Despite this information, the biochemical basis for megaloblastic is not fully
understood. This is especially true of the cobalamin-related neuropathy that can
occur independently of megaloblastic changes in hematopoietic cells. One
hypothesis for the cause of cobalamin neuropathy is that a defect exists in the
conversion of adenosyl-cobalamin-dependent conversion of methylmalonyl
coenzyme A to succinyl coenzyme A.

A hallmark of megaloblastic anemia is ineffective erythropoiesis, as evidenced by

erythroid hyperplasia in the bone marrow, a decreased peripheral reticulocyte count,
and an elevation in lactate dehydrogenase (LDH) and indirect bilirubin levels. The
pathogenesis of these findings is the intramedullary destruction of fragile and
abnormal megaloblastic erythroid precursors.

An understanding of the source of cobalamin and folate is important to understand

the pathogenesis of the development of megaloblastic. Dietary intake is the source
of cobalamin and folate because humans cannot synthesize these substances.
Cobalamin must be bound to intrinsic factor (IF), and this complex is taken up in the
terminal ileum. Once absorbed, cobalamin is bound to another protein,
transcobalamin II (TCII), and is transported to storage sites. Abnormalities in any of
these steps in cobalamin transport can lead to deficiencies in this substance.
Considerable amounts of cobalamin are accumulated in storage sites; this explains
why years elapse before cobalamin deficiency develops in patients who cannot take
up dietary cobalamin.

Although the processing and transport of ingested folate is complex, folate-induced

megaloblastic is rarely caused by abnormalities in transport but instead is most
often caused by dietary insufficiency. Folate deficiency can be caused by
malabsorption in patients with sprue. In contrast to cobalamin, very little folate is
stored; this explains why folate deficiency can occur within months of cessation of
folate ingestion.
 Megaloblastosis can also be caused by disorders in which cobalamin and folate
uptake and metabolism are not affected. Myeloproliferative syndromes and viral
infections (eg, HIV) can lead to megaloblastic by disrupting DNA synthesis.
Megaloblastosis can occur in patients who are on certain medications, including
many cancer chemotherapy drugs.

Dietary and pregnancy-related folate deficiencies are probably the most common
causes of megaloblastic anemias. However, current folate supplementation during
pregnancy and vitamin supplementation for elderly persons has resulted in a low
frequency of these forms of megaloblastic

Rare disorders:

i.Orotic aciduria: Deficiency of uridine monophosphate synthase leads to ↓ de novo

pyrimidine synthesis that is unresponsive to B12 and folate replacement.
ii.Methylmalonic acidemia: an inborn error of amino acid metabolism
a. Megaloblastic anemia caused by vitamin B12
❖ Causes
● Intrinsic factor deficiency (pernicious anemia)
● Malabsorption (Crohn disease, celiac disease, chronic pancreatitis, prior gastric
or ileal surgery)
● Vegan diet (vegan)
● Nitrous oxide abuse/toxicity
● Diphyllobothrium latum (fish tapeworm) infestation
● Drugs (immunosuppressants, isoniazid, metformin, colchicine, H2 blockers,
proton pump inhibitors)
● HIV
● Genetic disorders
❖ Clinical features
● B12 deficiency develops over years, whereas folate deficiency develops in weeks to
months.
● Depending on the degree of deficiency and time of onset, patients can be
asymptomatic.
● Signs and symptoms of anemia:
○ Fatigue
○ Shortness of breath
○ Tachycardia
○ Palpitations
 ○ Pallor
○ Jaundice
● GI symptoms (related to underlying GI conditions such as inflammatory bowel
disease):
○ Diarrhea
○ Bloating
○ Epigastric/abdominal pain
❖ Pathology
● Sources:
○ Animal products (meats, especially liver, dairy, eggs)
○ Fortified food
○ Not found in plants
● Absorption:
○ B12 in food is bound to protein and gets bound to salivary haptocorrins in the
stomach.
○ Absorption requires an acid environment (stomach) to be dissociated from
protein and pancreatic proteases to cleave off the haptocorrins.
○ Once unbound, B12 binds to intrinsic factors produced by the gastric parietal
cells.
○ B12–intrinsic factor complex is taken up in the ileum.
○ In the bloodstream, B12 is then endocytosed by cells. 11111
❖ Special manifestations
● Neurologic symptoms most commonly due to B12 deficiency:
○ Subacute combined degeneration (classic finding):
■ Dorsal column: vibration, proprioception (wide-based gait)
■ Lateral corticospinal tracts: spasticity
■ Dorsal spinocerebellar: ataxia
○ Neuropathy: tingling, numbness
○ Psychosis, depression, irritability
○ Cognitive impairment, forgetfulness
○ Dementia
● Additional findings in B12 deficiency:
○ Oral mucosa pathology (present in 50%–60% of patients):
■ Glossitis
■ Angular cheilitis
■ Recurrent oral ulcers
■ Diffuse erythematous mucositis/mucosal atrophy
■ Mouth soreness/burning sensation
○ Cutaneous hyperpigmentation
○ ↑ Risk of gastric cancer in pernicious anemia
● Folate deficiency: ↑ risk of neural tube defects (congenital anomaly)
b. Megaloblastic anemia caused by folic acid
❖ Causes
● Increased demand: pregnancy, hemolytic anemia, chronic dermatitis,
hemodialysis
● Alcoholism
● Dietary deficiency (restricted diets, countries without folate fortification of foods)
● Drugs (antimetabolites such as methotrexate, tetracyclines, penicillins,
nitrofurantoin, phenobarbital, phenytoin, trimethoprim)
● Intestinal dysfunction: Malabsorption occurs in surgery (gastric bypass).
❖ Pathology
○ Sources:
■ Plant products (especially dark green, leafy vegetables)
■ Animal products
○ Absorption:
■ Dependent on carrier systems
■ Absorbed in the jejunum (conjugase converts folate polyglutamate to
monoglutamate)
■ Subsequently reduced to dihydrofolate → tetrahydrofolate (THF) →
5,10-methylene THF → L-5-methyl-THF (predominant plasma form)
■ L-5-methyl-THF is taken up by cells.
● Both are water-soluble (↓ risk of overdose, as they are excreted in the urine).
● Vitamin deficiencies arise from conditions that affect intake and absorption and
that interfere with other essential factors (i.e., intrinsic factor, carrier system).
c. B12 and folic acid combined
a. Role of vitamin B12 and folic acid in DNA synthesis:
Dietary folate is absorbed in the intestine in the form of 5-methyl-tetrahydrofolate (THF).
Vitamin B12–dependent methionine synthetase converts 5-methyl-THF to THF. The
same process generates methionine from homocysteine and methionine converts to
S-adenosylmethionine or SAM (also necessary for DNA methylation).
The THF produced is converted to 5, 10-methylene-THF.
A methyl group is donated from methylene-THF to the 5-carbon of uridylate to form
thymidylate.
As a consequence of donating the methyl group, methylene-THF becomes dihydrofolate.
Dihydrofolate is reduced by reductase to re-generate tetrahydrofolate.

b. B12 and folic acid in DNA synthesis

➔ Both B12 and folic acid:
➔ Critical in the synthesis of nucleic acids
➔ Act as cofactors in the synthesis of thymidylate, the rate-limiting step in
DNA synthesis:
● Folate supplies the methyl groups (via methylene-THF).
● B12 is a cofactor in the reaction converting 5-methyl-THF to THF.
 ➔ Methionine and folic acid cycle with DNA synthesis:
➔ Dietary folate is absorbed in the intestine in the form of 5-methyl-THF.
➔ Vitamin B12–dependent methionine synthetase converts 5-methyl-THF to
THF.
● The same process generates methionine from homocysteine.
● Methionine converts to S-adenosylmethionine (SAM) (in the
methionine cycle), which is also necessary for DNA methylation.
➔ The THF produced is converted to methylene-THF.
➔ A methyl group is donated from methylene-THF to the 5-carbon of
uridylate to form thymidylate (deoxythymidine monophosphate).
➔ As a consequence of donating the methyl group, methylene-THF
becomes dihydrofolate.
➔ Dihydrofolate is reduced to regenerate tetrahydrofolate.
c. Consequences of defective DNA synthesis
Rapidly dividing cells in the body are most sensitive to impaired DNA synthesis
due to B12 and folate deficiency.
I. Impaired erythropoiesis:

a. Megaloblastic changes:
i. 0Slow nuclear division → immature, abnormal nuclei + normal cytoplasm
→ dyssynchrony between nuclear and cytoplasmic maturation
ii. Increased mitotic figures → giant metamyelocytes + finely stippled, lacy
nuclear chromatin pattern
b. Intramedullary hemolysis:
i.Caused by the premature death of abnormally developing erythroid
precursors
ii.Bone = The bone marrow becomes hypercellular owing to ongoing
apoptosis → ↑ hemolysis, ↓ reticulocyte count

II. Reduced DNA methylation:

Significant epigenetic modification → methyl groups added to DNA → result in

modification of gene expression. Results in defects in DNA repair and
fragmentation. Enhances the chance of translational errors including the
possibility of malignant transformation. Believed to contribute to neuronal
dysfunction in B12 deficiency:

↓ Methylation of lipids and proteins (i.e., myelin basic protein) in the

neurons
↓ Myelin basic protein contributes to demyelination.
 D. clinical presentation

❖General manifestations
● B12 deficiency develops over years, whereas folate deficiency develops in weeks to
months.
● Depending on the degree of deficiency and time of onset, patients can be
asymptomatic.
● Signs and symptoms of anemia:
○ Fatigue
○ Shortness of breath
○ Tachycardia
○ Palpitations
○ Pallor
○ Jaundice
● GI symptoms (related to underlying GI conditions such as inflammatory bowel
disease):
○ Diarrhea
○ Bloating
○ Epigastric/abdominal pain
❖Specific manifestations
I. Neurologic symptoms most commonly due to B12 deficiency:

➔ Subacute combined degeneration (classic finding):

● Dorsal column: vibration, proprioception (wide-based gait)
● Lateral corticospinal tracts: spasticity
● Dorsal spinocerebellar: ataxia
➔ Neuropathy: tingling, numbness
➔ Psychosis, depression, irritability
➔ Cognitive impairment, forgetfulness
➔ Dementia

II. Additional findings in B12 deficiency:

➔ Oral mucosa pathology (present in 50%–60% of patients):

● Glossitis
● Angular cheilitis
● Recurrent oral ulcers
● Diffuse erythematous mucositis/mucosal atrophy
● Mouth soreness/burning sensation
➔ Cutaneous hyperpigmentation
➔ ↑ Risk of gastric cancer in pernicious anemia

III. Folate deficiency: ↑ risk of neural tube defects (congenital anomaly)

E. Diagnosis

❖History
● Diet: vegan or vegetarian
● Medical and social history: look for autoimmune disorders, alcoholism
● Surgical history: gastric or ileal resection
● GI symptoms
● Neurologic symptoms
● Medications

❖Laboratory tests
● Hematologic tests:
○ ↓ Hemoglobin/hematocrit
○ MCV > 100
○ Likely decreased leukocyte and/or platelet count
○ Low reticulocyte count
● Cobalamin/B12 and folate levels :
○ Deficiency:
■ Low B12/cobalamin (normal, > 300 pg/mL)
■ Low folate levels (normal, > 4 ng/mL)
■ RBC folate can be an alternative for folate level (though not frequently
used).
○ For borderline levels of B12 (200–300 pg/mL) or folate (2–4 ng/mL), proceed with
obtaining homocysteine and methylmalonic acid (MMA) level:
■ Serum homocysteine level: elevated in both vitamin B12 and folate
deficiency
■ Serum MMA level: elevated in vitamin B12 deficiency
● Additional workup:
○ Pernicious anemia:
■ Anti-intrinsic factor antibodies: high specificity for pernicious anemia
■ Antiparietal cell antibodies: can be present in gastritis
■ ↑ Serum gastrin levels: not specific to pernicious anemia
○ Orotic aciduria: normal urine ammonia with ↑ orotic acid levels
 ● Peripheral-blood smear:
○ Macrocytosis
○ Marked RBC size variation: anisocytosis
○ Abnormal and variable RBC morphology: poikilocytosis
○ Hypersegmented neutrophils:
■ ≥ 1% of neutrophils with 6 or more nuclear lobes, OR
■ ≥ 5% of neutrophils with 5 or more nuclear lobes

❖Additional tests
● Bone marrow aspirate:
○ Not routinely done
○ If done, findings include:
■ Hypercellularity
■ Erythroid predominance with decreased myeloid to erythroid ratio
■ Nuclear cytoplasmic dyssynchrony with immature nuclei combined with
normal-appearing cytoplasm
■ Abnormal granulocytes: giant metamyelocytes and bands
■ Abnormally large megakaryocytes
■ In severe cases, dysplastic changes can be seen.
● Gastric biopsy:
○ Not necessary for the diagnosis of pernicious anemia
○ If done, will reveal chronic autoimmune atrophic gastritis
● Schilling test for pernicious anemia:
○ Infrequently used
○ Supplanted by serologic testing for parietal cell and intrinsic factor antibodies

F. management

❖ Principles
● Patients can be asymptomatic or deficiency can be an incidental finding.
● In the majority of cases, there is a gradual development of symptoms, so treatment
can be given over weeks.
● Intervention is needed urgently for the following:
○ Symptomatic anemia
○ Neurologic/neuropsychiatric manifestations (as effects can be irreversible)
○ Pregnancy (fetus is affected)
○ Neonates and infants (growth is affected)
❖ Treatment
● Vitamin B12:
○ Intramuscular/parenteral route for neurologic symptoms or in patients with
malabsorption or extensive gastric/bowel resections
○ Oral route for deficiency owing to low dietary intake or to continue replacement
from the parenteral route (if appropriate)
○ Watch for thrombocytosis and hypokalemia in severe cases.
● Folic acid:
○ Always check for concomitant B12 deficiency.
○ 1–5 mg/day, with the higher dose for pregnant women, alcoholics, patients on
antiepileptic medications
● Replacement can be lifelong if the underlying condition is permanent (i.e., gastric
bypass surgery).
● As anemia resolves, iron levels may become depleted; monitor and replace as
necessary.
● Treatment of other associated conditions:
○ Pernicious anemia: in addition to replacement, screen for gastric cancer
○ Uridine triacetate: for orotic aciduria

G. differential diagnosis

➔ Alcoholism and alcoholic liver disease: Alcohol is one of the top causes of
macrocytosis, usually due to a toxic effect directly on the RBCs (without damage to
DNA replication). Chronic alcoholism also leads to liver disease, ranging from a fatty
liver to cirrhosis. Management aims at alcohol abstinence for reversal at certain
stages; addressing contributing factors, such as viral infections or drugs; and
minimizing damage to the hepatocytes.
➔ Chronic liver disease: Cirrhosis is the late stage of hepatic necrosis and scarring.
Chronic cellular damage causes extensive distortion of the normal hepatic
architecture, which can lead to impairment of normal blood flow through the liver.
Chronic liver disease of diverse etiologies can cause macrocytic anemia owing to
effects in lipid composition of RBCs.
➔ Myelodysplastic anemia: a group of hematologic malignancies due to germline
mutations associated with one or more cytopenias, including macrocytic anemia:
Dysplastic changes in the bone marrow biopsy, such as hyposegmented
granulocytes and blasts, are seen. Cytogenetic analysis identifying mutations linked
to myelodysplastic disease helps establish the diagnosis.
➔ Aplastic anemia: a disorder of bone marrow failure characterized by absence of
erythroid precursors due to exposure to drugs, radiation, chemicals, viruses,
autoimmune disease, or genetic factors (hereditary or acquired): The bone marrow is
hypocellular, with < 30% cellularity. Some cytopenias involve ≥ 2 cell lines.
➔ Hypothyroidism: a condition characterized by a deficiency of thyroid hormones:
Iodine deficiency is the most common cause worldwide, but Hashimoto disease
(autoimmune thyroiditis) is the leading etiology in non–iodine-deficient regions.
Features of acquired hypothyroidism include fatigue, bradycardia, cold intolerance,
and exertional dyspnea. Diagnosis is by thyroid function tests. Elevated
thyroid-stimulating hormone and a low free thyroxine (T4) are noted. Treatment is
with synthetic T4. Macrocytic anemia is seen in 55% of patients with hypothyroidism.
 3. Sickle cell anemia
a. Introduction
Sickle cell disease (SCD) is a group of inherited red blood cell disorders. If you have
SCD, there is a problem with your hemoglobin. Hemoglobin is a protein in red blood
cells that carries oxygen throughout the body. With SCD, the hemoglobin forms into
stiff rods within the red blood cells. This changes the shape of the red blood cells. The
cells are supposed to be disc-shaped, but this changes them into a crescent, or
sickle, shape.

The sickle-shaped cells are not flexible and cannot change shape easily. Many of
them burst apart as they move through your blood vessels. The sickle cells usually
only last 10 to 20 days, instead of the normal 90 to 120 days. Your body may have
trouble making enough new cells to replace the ones that you lost. Because of this,
you may not have enough red blood cells. This is a condition called anemia, and it
can make you feel tired.

The sickle-shaped cells can also stick to vessel walls, causing a blockage that slows
or stops the flow of blood. When this happens, oxygen can't reach nearby tissues. The
lack of oxygen can cause attacks of sudden, severe pain, called pain crises. These
attacks can occur without warning. If you get one, you might need to go to the
hospital for treatment.

b. Causes of sickle cell anemia

The cause of SCD is a defective gene, called a sickle cell gene. People with the disease
are born with two sickle cell genes, one from each parent.

If you are born with one sickle cell gene, it's called sickle cell trait. People with sickle
cell traits are generally healthy, but they can pass the defective gene on to their
children.

c. Symptoms of sickle cell anemia

People with SCD start to have signs of the disease during the first year of life, usually
around 5 months of age. Early symptoms of SCD may include

● Painful swelling of the hands and feet

● Fatigue or fussiness from anemia
● A yellowish color of the skin (jaundice) or the whites of the eyes (icterus)

The effects of SCD vary from person to person and can change over time. Most of the
signs and symptoms of SCD are related to complications of the disease. They may
include severe pain, anemia, organ damage, and infections.

d. Diagnosis of sickle cell anemia

A blood test can show if you have SCD or sickle cell trait. All states now test newborns
as part of their screening programs, so treatment can begin early.

People who are thinking about having children can have the test to find out how likely
it is that their children will have SCD.
 Doctors can also diagnose SCD before a baby is born. That test uses a sample of
amniotic fluid (the liquid in the sac surrounding the baby) or tissue taken from the
placenta (the organ that brings oxygen and nutrients to the baby).

e. Treatment of sickle cell anemia

The only cure for SCD is bone marrow or stem cell transplantation. Because these
transplants are risky and can have serious side effects, they are usually only used in
children with severe SCD. For the transplant to work, the bone marrow must be a close
match. Usually, the best donor is a brother or sister.

There are treatments that can help relieve symptoms, lessen complications, and
prolong life:

● Antibiotics to try to prevent infections in younger children

● Pain relievers for acute or chronic pain
● Hydroxyurea, a medicine that has been shown to reduce or prevent several SCD
complications. It increases the amount of fetal hemoglobin in the blood. This
medicine is not right for everyone; talk to your health care provider about
whether you should take it. This medicine is not safe during pregnancy.
● Childhood vaccinations to prevent infections
● Blood transfusions for severe anemia. If you have had some serious
complications, such as a stroke, you may have transfusions to prevent more
complications.

There are other treatments for specific complications.

To stay as healthy as possible, make sure that you get regular medical care, live a
healthy lifestyle, and avoid situations that may set off a pain crisis.

4. Thalassemia
Thalassemia is an inherited blood disorder wherein the body produces an
inadequate amount of hemoglobin. Hemoglobin is a protein molecule that carries
oxygen in the red blood cells. This disorder results in the extreme destruction of
red blood cells that leads to anemia. Anemia is a condition in which the
hemoglobin or red blood cells are less than the normal count.
Thalassemias are a group of hereditary hemoglobin disorders characterized by
mutations on the α- or β-globin chains (resulting in alpha or beta-thalassemia).
Thalassemias can be further classified according to the specific genotype: the
α-chain is coded by four alleles, resulting in four possible variants depending on
the number of alleles affected, while the β-chain is coded by two alleles, resulting
in two possible variants. The number of alleles affected is directly related to the
severity of the disease (minor/intermedia/major).
It is an inherited disease that is mainly caused due to abnormal hemoglobin
synthesis. It is transferred by one of the parents who is a carrier of this disease
due to either deletion of particular key gene fragments or a genetic mutation.
 Mild thalassemia requires no treatment, but acute thalassemia might require
regular blood transfusions.
The key feature in all forms of thalassemia is microcytic hypochromic
Anemia (which may be very mild in minor forms), but more severe forms may also
manifest with hemolysis, splenomegaly, delay in growth and development, and
skeletal deformities. The diagnostic workup for suspected thalassemia includes a
blood smear, hemoglobin electrophoresis, high-performance liquid
chromatography (HPLC), and, possibly, genetic testing. Minor forms of thalassemia
usually require no treatment, while patients with thalassemia intermedia/major
typically require regular blood transfusions and management of disease and
treatment-related complications (e.g., chelating agent for transfusion-mediated
iron overload.
❖ Types of Thalassemia
There are two types of thalassemia:
● Alpha-thalassemia – A disorder in which one of the genes of alpha-globin has
a mutation or abnormality.
● Beta-thalassemia – The genes of beta-globin are abnormal.
❖ Epidemiology
● Beta thalassemia is most commonly seen in people of Mediterranean descent
● Alpha thalassemia: most commonly seen in people of Asian and African descent
● Thalassemia provides partial resistance against malaria
a. Causes of thalassemia
It develops when there is some abnormality in any one of the genes that are
involved in the production of hemoglobin and this defect is inherited from the
parents. If any of the parents have thalassemia, the baby is more likely to develop
this disease so-called thalassemia minor. If both the parents suffer from this
disease, you are more likely to get the disease.
There are no symptoms at an early stage but likely to be a disease carrier. It is the
most common disease in people of Asia, Africa, the Middle East, Turkey, and
Greece.
b. Alpha-thalassemia
It consists of two major forms namely, Hydrops fetalis or Haemoglobin H disease.
Alpha-thalassemia is usually due to deletion of at least one out of four existing alleles.
The alpha-globin gene cluster is located on chromosome 16.
Haemoglobin H can be responsible for bone complexities. The forehead, cheeks, and
jaw may overgrow. Moreover, hemoglobin H can cause:
● An intensely enlarged spleen.
● Malnourishment.

In a normal cell, the α-globin chains are coded by a total of four alleles. Thus,
there are four forms of the disease. The severity of alpha thalassemia
depends on the number of defective α-globin alleles.
Silent carrier (minima form): one defective allele (-α/αα)
 Alpha thalassemia trait (minor form)

i.Two defective alleles (-α/-α or --/αα)

ii.Cis-deletion is common amongst Asian populations, whereas trans-deletions
are more common in African populations
iii.Children of parents with a two-gene deletion in cis are at higher risk of
developing Hb Bart.

Hemoglobin H disease (intermedia form): three defective alleles (--/-α) →

results in excessive production of pathologically altered HbH
Hemoglobin Bart disease (major form): four defective alleles (--/-‑) → results in
excessive production of pathologically altered Hb Bart (consists of four
γ-chains (γ-tetramers)
c. Beta-thalassemia
Beta thalassemia occurs in two different forms namely thalassemia intermedia and
thalassemia major.
Beta-thalassemia is usually due to point mutation in promoter sequences or
splicing sites. Beta-globin locus = short arm of chromosome 11.
Beta thalassemia is a hemoglobinopathy characterized by impaired production of
one or both beta chains of hemoglobin.
Thalassemia symptoms appear generally before a child’s second year of age and
severe anemia concerned with this condition can be fatal. Some of the major signs
of thalassemia major include:
● Paleness
● Jaundice
● Fussiness
● Poor appetite

This kind of thalassemia is so serious that it needs frequent blood transfusions.

Thalassemia intermedia is a less serious kind of beta-thalassemia and does not
require the patient to go through blood transfusions.
Beta-Thalassemia trait is found in individuals where there is only one HBB gene
mutation in each cell possesses mild anemia.
In a normal cell, the β-globin chains are coded by a total of two allelesThus, there are
two main forms of the disease.
● Beta thalassemia minor (trait): one defective allele
● Beta thalassemia major (Cooley anemia): two defective alleles
● Sickle cell beta thalassemia: a combination of one defective β-globin allele and one
defective HbS allele
● Hemoglobin E/beta thalassemia: a combination of one allele with a hemoglobin E
(HbE) variant and one defective β-globin allele. Produces a highly heterogeneous
clinical spectrum, and in severe cases patients present with features of beta
thalassemia major
● Hemoglobin E disease: a condition characterized by homozygosity to the HbE
variant. Patients can present with mild features resembling beta thalassemia minor
(i.e., mild anemia).
d. Treatment for thalassemia
The treatment depends on the type and severity of the disease. The doctor
provides a course of treatment that suits best for a particular case.
Some of the treatments, which are opted for maximum cases include:
● Bone marrow transplant (BMT)
● Supplements and Medications
● Blood transfusions

Few precautions are prescribed by the doctors that include not taking
supplements or vitamins and minerals containing iron. This is true when there is
a need for blood transfusions. Patients who go through blood transfusions
obtain extra iron which a body cannot lose.
If you are receiving a blood transfusion, you may need chelation therapy. It
includes taking a chemical injection that combines with other heavy metals and
iron. This helps eliminate extra iron from the body.
e. Prevention of thalassemia
Thalassemia cannot be prevented since it is a genetically inherited disorder.
However, these disorders can be detected during prenatal tests before birth.
Also, genetic counseling helps to detect whether people have altered or missing
hemoglobin genes that cause thalassemia.
f. Pathophysiology
Anemia results from a combination of inefficient erythropoiesis and increased
hemolysis. The degree to which both mechanisms contribute to the severity of the
disease depends on a patient's exact genotype.
● Inefficient erythropoiesis → anemia
○ Beta thalassemia minor and major: faulty β-globin chain synthesis → ↓
β-chains→ ↑ γ-,δ-chains → ↑ HbF and ↑ HbA2.
○ HbF protects infants up to the age of 6 months, after which HbF production
declines and symptoms of anemia appear.
○ Alpha thalassemia intermedia (HbH disease) and alpha thalassemia major
(Bart disease): faulty α-globin chain synthesis → ↓ α-chains → impaired
pairing of α-chains with β-chains and γ-chains→ ↑ free β-, γ-chains → ↑ HbH, ↑
Hb-Bart's
○ In minor and minima forms, production of the affected chain is reduced, but
enough is produced to prevent severe anemia.
● Increased hemolysis: One of the chains (either α or β) is reduced → compensatory
overproduction of other chains → excess globin chains precipitate and form
inclusions within RBCs → erythrocyte instability with hemolysis
● Anemia → ↑ erythropoietin → bone marrow hyperplasia and skeletal deformities
g. Clinical features
I. beta-thalassemia
● Minor variant: unremarkable symptoms (low risk of hemolysis,rarely splenomegaly)
● Major variant
○ Severe hemolytic anemia that often requires transfusions; → secondary iron
overload due to hemolysis, transfusion, or both → secondary
hemochromatosis
○ Hepatosplenomegaly
○ Growth retardation
○ Skeletal deformities (high forehead, prominent zygomatic bones, and maxilla)
○ Transient aplastic crisis secondary to infection with parvovirus B19)
● Sickle cell beta thalassemia
○ Features of sickle cell disease
○ Severity depends on the amount of β-globin synthesis.

II. alpha-thalassemia
● Silent carrier: asymptomatic
● Alpha thalassemia trait = mild hemolytic anemia with normal RBC and RDW
● Hemoglobin H disease
○ Jaundice and anemia at birth
○ Chronic hemolytic anemia that may require transfusions → secondary iron
overload due to hemolysis, transfusion, or both → secondary
hemochromatosis
○ Hepatosplenomegaly
○ Skeletal deformities (less common)
○ Compared to thalassemia beta, symptoms in adults are generally less severe.
● Hb-Bart's hydrops fetalis syndrome (most severe variant of alpha thalassemia)
○ Intrauterine ascites and hydrops fetalis; , severe hepatosplenomegaly, and
often cardiac and skeletal anomalies
○ Incompatible with life (death in utero or shortly after birth)