HEMATOLOGY 1

MIDTERMS: CHAPTER 18 - Anemias Caused by Defects of DNA Metabolism

CHAPTER 18 - Anemias Caused by Defects of DNA
Metabolism
General Terms:
• Impaired deoxyribonucleic acid (DNA) metabolism
causes systemic effects by impairing production of
all rapidly dividing cells of the body
• These are chiefly the cells of the skin, the epithelium
of the gastrointestinal tract, and the hematopoietic
tissues.
• Because these all must be replenished throughout
life, any impairment of cell production is evident in
these tissues first.
ETIOLOGY:
• The root cause of megaloblastic anemia is impaired
DNA synthesis.
• Megaloblastic anemia is one example of a
macrocytic anemia.

Physiologic Roles of Vitamin B12 and Folate

• The role of VitB12 and Folate When either folate or
vitamin B12 is missing, thymidine nucleotide
production for DNA synthesis is impaired.
• In the absence of vitamin B12, the impaired activity
of methylmalonyl CoA mutase leads to a high level
of serum methylmalonic acid (MMA)
o which is useful for the diagnosis of
vitamin B12 deficiency
• Methylcobalamin is synthesized through reduction
and methylation of vitamin B12.
• To distinguish between the intrinsic factor and
cobalamin. Cobalamin and transcobalamin II will
bind in that order for the cobalamin to be delivered
to the bloodstream or plasma.
• FOLATE:
Defect in Megaloblastic Anemia Caused By Deficiency in
o Folate is the general term used for any
Folate and Vitamin B12
form of the vitamin folic acid
• When either folate or vitamin B12 is deficient,
o Folic acid is the synthetic form in
thymidine nucleotide production for DNA synthesis
supplements and fortified food.
is impaired.
o Folates consist of a pteridine ring attached
to Para aminobenzoate with one or more • Folate deficiency has the more direct effect,
glutamate residues ultimately preventing the methylation of dUMP.
o The function of folate is to transfer • The effect of vitamin B12 deficiency is more indirect,
carbon units in the form of methyl preventing the production of THF from 5-methyl
groups from donors to receptors. THF.
o In this capacity folate plays an important o This constitutes what has been called
role in the metabolism of amino acids and the folate trap
nucleotides • In addition, when either folate or vitamin B12 is
o Deficiency of the vitamin leads to impaired deficient, homocysteine accumulates because
cell replication and other metabolic methionine synthase is unable to convert it to
alterations. methionine
o Folate has an important role in DNA • The DNA repair process can remove the uridine, but
synthesis. without available thymidine to replace it, the repair
process is unsuccessful.
• Cells that survive continue the abnormal maturation
with a fewer number of red blood cells (RBCs)
released into the circulation.

Alexis Jade S. Gamboa MT-301

 HEMATOLOGY 1
MIDTERMS: CHAPTER 18 - Anemias Caused by Defects of DNA Metabolism
and are refractive to therapy with vitamin B12 or folic
acid.

SYSTEMIC MANIFESTATIONS OF FOLATE AND

VITAMIN B12 DEFICIENCY
• When DNA synthesis and subsequent cell division
are impaired by lack of folate or vitamin B12
o megaloblastic anemia and its systemic
manifestations develop.
• With either vitamin deficiency, patients may
experience general symptoms related to anemia
(fatigue, weakness, and shortness of breath) and
symptoms related to the alimentary tract.
o This abnormal blood cell development is
• Loss of epithelium on the tongue results in a
called ineffective hematopoiesis
smooth surface and soreness (glossitis)
o The dependency of DNA production on
o result in gastritis, nausea, or
folate has been used in cancer
constipation
chemotherapy
• After dietary deficiency or malabsorption begins, it
• The nuclear changes seen in the megaloblastic
takes a few years to develop a vitamin B12
erythroid precursors are related to cell cycle delay,
deficiency.
prolonged resting phase, and arrest in nuclear
• but only a few months to develop a folate
maturation.
deficiency
• Ribonucleic acid (RNA) function is not affected
• In vitamin B12 deficiency, neurologic symptoms
by vitamin B12 or folate deficiency because RNA
may be pronounced and may even occur in the
contains uracil instead of thymidine
absence of anemia
nucleotides,
o These include memory loss, numbness
• The slower maturation rate of the nucleus compared
and tingling in toes and fingers, loss of
with the cytoplasm is called nuclear-cytoplasmic
balance, and further impairment of walking
asynchrony
by loss of vibratory sense, especially in the
• Because ineffective hematopoiesis affects all three
lower limbs
blood cell lineages, pancytopenia is also evident,
o Neuropsychiatric symptoms may also be
with certain distinctive cellular changes
present, including personality changes
and psychosis
Other Causes of Megaloblastosis
▪ These symptoms seem to be the
• Vitamin B12 and folate deficiency are not the only
result of demyelination of the
causes of megaloblastic erythroid precursors.
spinal cord and peripheral nerves
• Dysplastic erythroid precursors in
• folate deficiency was believed to be more benign
myelodysplastic syndrome (MDS) can also have
clinically than vitamin B12 deficiency
megaloblastoid feature.
• Later research suggested that low levels of folate
• nuclear-cytoplasmic asynchrony and megaloblastic
and the resulting high homocysteine levels were risk
erythroid precursors may be seen in congenital
factors for cardiovascular disease
dyserythropoietic anemia (CDA) types I and III
o Several studies suggest that high folate
o In CDA I, internuclear chromatin bridging
levels provide a cardioprotective effect in
of erythroid precursors or binucleated
diabetic patients and certain ethnic
forms are observed
populations.
o in CDA III, giant multinucleated
• The evidence at this time is unclear as to whether
erythroblasts are present.
persistent suboptimal folate status may have a
• acute erythroid leukemia
significant long-term health impact.
o In this condition the erythroblasts are
o In addition, there is evidence of
macrocytic, and the immature appearance
depression, peripheral neuropathy, and
of the nuclear chromatin is similar to the
psychosis related to folate deficiency.
more open appearance of the chromatin in
o Folate levels appear to influence the
megaloblasts
effectiveness of treatments for depression.
• they are due to acquired or inherited mutations in
o Folate deficiency during pregnancy can
progenitor cells or interference with DNA synthesis
result in impaired formation of the fetal

Alexis Jade S. Gamboa MT-301

 HEMATOLOGY 1
MIDTERMS: CHAPTER 18 - Anemias Caused by Defects of DNA Metabolism
nervous system, resulting in neural tube for this and the result is macrocytosis with
defects such as spina bifida. frank megaloblastic anemia.
• Fortification of the US food supply with folic acid in o Because folate deficiency results in
grain and cereal products was mandated by the inhibition of cell replication, several
Food and Drug Administration in 1998. anticancer drugs, including methotrexate,
are folate inhibitors
CAUSES OF VITAMIN DEFICIENCIES
Folate Deficiency
• Inadequate Intake
o Folate is synthesized by microorganisms
and higher plants.
o Folate is ubiquitous in foods, but a
generally poor diet can result in deficiency.
o Good sources of folate include
▪ leafy green vegetables, dried
beans, liver, beef, fortified
breakfast cereals, and some
fruits, especially oranges
• Increased Need
o Increased need for folate occurs during
pregnancy and lactation when the mother
must supply her own needs plus those of
the fetus or infant • Excessive Loss of Folate
o Infants and children also have increased o Physiologic loss of folate occurs through
need for folate during growth the kidney.
o The amount is small and not a cause of
• Impaired Absorption
deficiency
o Food folates must be hydrolyzed in the gut
o Patients undergoing renal dialysis lose
before absorption in the small intestine.
folate in the dialysate
▪ only 50% of what is ingested is
o supplemental folic acid is routinely
available for absorption
provided to these individuals to prevent
o A rare autosomal recessive deficiency of a
megaloblastic anemia
folate transporter protein (PCFT) severely
Vitamin B12 Deficiency
decreases intestinal absorption of folate.
o Folate absorption may also be impaired by • Inadequate Intake
intestinal disease, especially sprue and o Although true dietary deficiency of vitamin
celiac disease B12 is rare
▪ Sprue is characterized by o This condition is possible for strict
weakness, weight loss, and vegetarians
steatorrhea (fat in the feces) o it is not available from vegetable sources
▪ Celiac disease (nontropical o The best dietary sources are animal
sprue) has been traced to products such as liver, dairy products, fish,
intolerance of gluten in some shellfish, and eggs
grains o In contrast to the heat-labile folate, vitamin
B12 is not destroyed by cooking.
• and can be controlled by
eliminating wheat, • Increased Need
barley, and rye products o Increased need for vitamin B12 occurs
from the diet. during pregnancy, lactation, and growth.
o Surgical resection of the small intestine ▪ Because of the vigorous cell
and inflammatory bowel disease can also replication, what would otherwise
decrease folate absorption be a diet adequate in vitamin B12
can become inadequate during
• Impaired Use of Folate
these periods.
o Numerous drugs decrease absorption of
folic acid or impair folate metabolism • Impaired Absorption
o Antineoplasic, antibacterial, and o Vitamin B12 in food is released from food
antiseizure agents are particularly known proteins primarily in the acid environment
of the stomach, aided by pepsin, and is

Alexis Jade S. Gamboa MT-301

 HEMATOLOGY 1
MIDTERMS: CHAPTER 18 - Anemias Caused by Defects of DNA Metabolism
subsequently bound by a specific salivary of parietal cells in Helicobacter
protein, haptocorrin, also known as R pylori infection
protein or transcobalamin I. • Pernicious anemia
o In the small intestine, vitamin B12 is o Pernicious anemia is an autoimmune
released from haptocorrin by the action disorder characterized by impaired
of pancreatic proteases absorption of vitamin B12 because of an
o It is then bound by intrinsic factor, which is intrinsic factor deficiency.
produced by gastric parietal cells. Vitamin o This condition is called pernicious anemia
B12 binding to intrinsic factor is required because the disease was fatal before its
for absorption by ileal enterocytes that cause was discovered.
possess receptors for the complex. o Pathologic CD4 T cells inappropriately
o These receptors are cubilin-amnionless recognize and initiate an autoimmune
complex: response against the H1/K1
▪ cubam, which binds the ▪ A chronic inflammatory infiltration
vitamin B12-intrinsic factor follows, which extends into the
complex wall of the stomach
▪ megalin, a membrane o The loss of H+ production in the stomach
transport protein constitutes achlorhydria
o The vitamin B12- transcobalamin complex, o Low gastric acidity was previously an
termed holotranscobalamin (holoTC) important diagnostic criterion for
• The absorption of vitamin B12 can be impaired by: pernicious anemia
o (1) failure to separate vitamin B12 from o Serum gastrin levels can be markedly
food proteins in the stomach elevated as a result of the gastric
▪ A condition known as food- achlorhydria
cobalamin malabsorption is o The absence of intrinsic factor can also be
characterized by hypochlorhydria detected using the Schilling test
▪ Food-cobalamin malabsorption is o Another feature of the autoimmune
caused primarily by the response in pernicious anemia is the
reduced gastric acidity in production of antibodies to intrinsic factor
atrophic gastritis or atrophy of and gastric parietal cells that are
the stomach lining that often detectable in serum
occurs with increasing age o Parietal cell antibodies are detectable in
▪ It also occurs with gastric bypass the serum of 50% to 90% of patients with
pernicious anemia
surgery and the long-term use of
• Other causes of lack of intrinsic factor
histamine type 2 receptor
o A lack of intrinsic factor may also be
blockers and proton pump
related to H. pylori infection
inhibitors o colonization of the gastric mucosa with H.
o (2) failure to separate vitamin B12 from pylori progresses until the parietal cells
haptocorrin in the intestine are entirely destroyed.
▪ Lack of gastric acidity or lack o In addition, partial or total gastrectomy,
of trypsin which results in removal of intrinsic factor-
▪ result of chronic pancreatic producing parietal cells, invariably leads
disease can prevent vitamin B12 to vitamin B12 deficiency.
absorption because the vitamin o (4) malabsorption
remains bound to haptocorrin in ▪ caused by the same conditions
the intestine and unavailable to interfering with folate absorption,
intrinsic factor such as celiac disease, tropical
o (3) lack of intrinsic factor sprue, and inflammatory bowel
▪ Lack of intrinsic factor constitutes disease.
o (4.5) Inherited errors of vitamin B12
a significant cause of impaired
absorption and transport.
vitamin B12 absorption.
▪ Imerslund-Gräsbeck
▪ It is most commonly a result of
syndrome is a rare autosomal
autoimmune disease recessive condition caused by
▪ can also result from hereditary mutations in the genes for either
intrinsic factor deficiency or loss cubilin or amnionless.

Alexis Jade S. Gamboa MT-301

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MIDTERMS: CHAPTER 18 - Anemias Caused by Defects of DNA Metabolism
▪ Transcobalamin deficiency is o Additional morphologic changes may
another rare autosomal include the presence of teardrop cells
recessive condition resulting in a (dacryocytes), RBC fragments, and
deficiency of physiologically microspherocytes.
available vitamin B12 o The presence of schistocytes sometimes
o (5) competition for available vitamin leads to a paradoxically lower MCV than is
B12. seen in less severe cases.
▪ come from intestinal
o Nucleated RBCs, Howell-Jolly bodies,
organisms.
basophilic stippling, and Cabot rings may
▪ The fish tapeworm
also be observed.
Diphyllobothrium latum is able
to split vitamin B12 from • White blood cell (WBC) Manual Differential
intrinsic factor, rendering the Count
vitamin unavailable for host o Hypersegmentation of neutrophils is
absorption. essentially pathognomonic for
▪ Also, blind loops megaloblastic anemia.
• portions of the o Hypersegmented neutrophils noted in the
intestines that are WBC differential report are a significant
stenotic as a result of finding and require a reporting rule that can
surgery or be applied consistently because even
inflammation, can healthy individuals may have an
become overgrown with occasional one
intestinal bacteria that o One such rule is to report
compete effectively with
hypersegmentation when there are at least
the host for available
5 five-lobed neutrophils per 100 WBCs.
vitamin B12.
o Some laboratories perform a lobe count on
▪ In both these cases, the host is
unable to absorb sufficient 100 neutrophils and then calculate the
vitamin B12, and megaloblastic mean.
anemia results. o In megaloblastic anemia, the mean lobe
count should be greater than 3.4
LABORATORY DIAGNOSIS o a search for neutrophil hypersegmentation
Screening Tests on a peripheral blood film constitutes an
Five tests used to screen for megaloblastic anemia are inexpensive yet sensitive screening
• Complete blood count (CBC), and reticulocyte test for megaloblastic anemia.
count • Serum bilirubin, and lactate dehydrogenase.
o Slight macrocytosis often is the earliest o Although generally considered a nutritional
sign of megaloblastic anemia. anemia, megaloblastic anemia is in one
o expected to have decreased hemoglobin sense a hemolytic anemia.
and hematocrit values, pancytopenia, and o erythroid progenitors and precursors die
reticulocytopenia during division in bone marrow, many
o Hemoglobin values of less than 7 or 8 RBCs never enter the circulation
g/dL are not unusual (ineffective hematopoiesis)
o The mean cell volume (MCV) is usually o The usual signs of hemolysis are evident
100 to 150 fL in the serum, including an elevation in
o The mean cell hemoglobin (MCH) is levels of total and indirect bilirubin and
elevated by the increased volume of the lactate dehydrogenase (predominantly
cells, RBC derived)
o mean cell hemoglobin concentration SPECIFIC DIAGNOSIC TESTS
(MCHC) is usually within the reference Bone Marrow Examination
interval • Modern tests for vitamin deficiencies and
o The red blood cell distribution width (RDW) autoimmune antibodies have made bone marrow
is also elevated. examination an infrequently used diagnostic test for
o The characteristic morphologic findings of megaloblastic anemia.
megaloblastic anemia in the peripheral • Megaloblastic, in contrast to macrocytic, anemia
blood include oval macrocytes (enlarged refers to specific morphologic changes in the
oval RBCs and hypersegmented developing erythroid precursors
neutrophils with six or more lobes

Alexis Jade S. Gamboa MT-301

 HEMATOLOGY 1
MIDTERMS: CHAPTER 18 - Anemias Caused by Defects of DNA Metabolism
• The cells are characterized by a nuclear- Assays for Folate, Vitamin B12, Methylmalonic Acid and
cytoplasmic asynchrony in which the cytoplasm Homocysteine
matures as expected with increasing pinkness as
• Although bone marrow aspiration is confirmatory
hemoglobin accumulates
for megaloblastosis
• The nucleus lags behind, however, appearing
• the confirmation of megaloblastic morphology in
younger than expected for the degree of maturity of the marrow does not identify its cause.
the cytoplasm. • Tests for serum levels of folate and vitamin B12
• Overall, the bone marrow is hypercellular, with a are readily available using immunoassay
reduced myeloid-to-erythroid ratio of about 1:1 by • serum vitamin B12 may also be assayed by
virtue of the increased erythropoietic activity competitive binding chemiluminescence.
• Hematopoiesis is ineffective, however, and • Some laboratories conduct a reflexive assay for
although cell production in the bone marrow is MMA if vitamin B12 levels are low.
increased, the apoptosis of hematopoietic cells in • Methylmalonic acid is assayed by gas
the marrow results in peripheral pancytopenia. chromatography-tandem mass spectrometry
• Homocysteine levels are affected by deficiencies in
either folate or vitamin B12. 5-Methyl THF donates
a methyl group to homocysteine in the generation
of methionine.
• Homocysteine levels are also elevated in patients
with renal failure and dehydration.

Gastric Analysis and Serum Gastrin

• Gastric analysis may be used to confirm

achlorhydria, an expected finding in pernicious
anemia.
• Achlorhydria occurs in other conditions, however,
including natural aging.
• The H+ concentration is determined by pH
measurement.
• As a result of the gastric achlorhydria, serum
gastrin levels can be markedly elevated
o Serum gastrin is measured by
immunoassay, including
chemiluminescent immunometric assays.
(Giant Band) in megaloblastic anemia Antibody Assays

• Antibodies to intrinsic factor and parietal cells can

be detected in the serum of most patients with
pernicious anemia.
• Various immunoassays can detect intrinsic factor-
blocking antibodies
• parietal cell antibodies can be detected by indirect
fluorescent antibody techniques or enzyme-linked
immunosorbent assays
• Anti-intrinsic factor antibodies are highly
specific and confirmatory for pernicious
anemia

Holotranscobalamin Assay

• Holotranscobalamin is the metabolically active

form of vitamin B12.
• not suitable for use in clinical laboratories
• recommend its use in screening for metabolic
vitamin B12 deficiency
• Megakaryocytes do not show consistent changes
in megaloblastic anemia.

Alexis Jade S. Gamboa MT-301

 HEMATOLOGY 1
MIDTERMS: CHAPTER 18 - Anemias Caused by Defects of DNA Metabolism
Stool Analysis for Parasites

• a stool analysis for eggs or proglottids of the fish

tapeworm D. latum may be part of the diagnostic
workup.

MACROCYTIC NONMEGALOBLASTIC ANEMIAS

• The macrocytic nonmegaloblastic anemias are
macrocytic anemias in which DNA synthesis is
unimpaired.
• The macrocytosis tends to be mild; the MCV usually
ranges from 100 to 110 fL and rarely exceeds
120 fL
• lack hypersegmented neutrophils and oval
macrocytes in the peripheral blood and
megaloblasts in the bone marrow.
• Macrocytosis may be physiologically normal, as in
the newborn or the result of a pathologic condition:
o as in liver
o disease, chronic alcoholism, or bone
marrow failure
• Reticulocytosis is a common cause of
macrocytosis
TREATMENT
• Vitamin B12 may be administered intramuscularly to
treat pernicious anemia to bypass the need for
intrinsic factor
• High-dose oral vitamin B12 treatment is increasingly
popular in the treatment of pernicious anemia.
• Regardless of the treatment modality, those with
pernicious anemia or malabsorption must have
lifelong vitamin replacement therapy
• Folic acid can be administered orally

Alexis Jade S. Gamboa MT-301

 HEMATOLOGY 1
MIDTERMS: CHAPTER 18 - Anemias Caused by Defects of DNA Metabolism
can be caused by intestinal disorders, such as
• Iron is often supplemented concurrently to support sprue, celiac disease, and inflammatory bowel
the rapid cell production that accompanies effective disease. Competition for vitamin B12 can
treatment develop from an intestinal parasite
• Bone marrow morphology will begin to revert to a (Diphyllobothrium latum) or bacteria in
normoblastic appearance within a few hours intestinal blind loops
• A substantial reticulocyte response is apparent • Pernicious anemia is vitamin B12 deficiency
at about 1 week resulting from an autoimmune disease that
• hemoglobin increasing toward normal levels in causes destruction of gastric parietal cells. H+
about 3 weeks and intrinsic factor secretion is lost. Antibodies
• Hypersegmented neutrophils disappear from the to parietal cells or intrinsic factor, or both, are
peripheral blood within 2 weeks detectable in the serum.
• Thus, with proper treatment, hematologic
parameters may return to normal within 3 to 6
weeks and correction of the megaloblastic
anemia may occur in 6 to 8 weeks.

QUICK SUMMARY (BOOK) Must Remember!

• Impaired DNA synthesis affects all rapidly
dividing cells of the body, including those in
skin, gastrointestinal tract, and bone marrow.
The effect on hematologic cells results in
megaloblastic anemia
• Vitamin B12 deficiency is associated with
peripheral neuropathies and neuropsychiatric
abnormalities as a result of demyelinization of
nerves in the peripheral and central nervous
system.
• Impaired absorption of vitamin B12 can be
caused by several mechanisms. Decrease in
gastric acid production or lack of trypsin in the
intestine causes vitamin B12 to be excreted in
the stool rather than absorbed. Malabsorption

Alexis Jade S. Gamboa MT-301