Clinically oriented presentation 2014

1. Megaloblastic
Anemias
BY; DOCTOR IMRAN SHAHZAD ANJUM,MD
LATIN AMERICAN SCHOOL OF MEDICINE HAVANA CUBA

2. Concept
 An anemia in which there is a predominant number of
megaloblasstic erthyroblasts, and relatively few normoblasts, among
the hyperplastic erythroid cells in bone marrow (as in pernicious
anemia).

3. Causes of megaloblastic anemia

4. Cobalamin (vitamin B12)
 In nature mainly in 2-deoxyadenosyl (ado) form that is cofactor for
enzyme methylmalonyl coenzyme A mutase and other is
methylcobalamin in human plasma and in cell cytoplasm that is a
cofactor for methionine synthase.
 It is solely synthesized by microorganisms and the only source for
humans is food of animal origin like meat, fish and dairy products.
 Adult daily losses (mainly in urine and feces) are 1-3 ug and as body
doesnot have the ability to degrade body store, so daily requirements
are also 1-3 ug.
 Body stores are of the order of 2-3 mg, sufficient for 3-4 years if supplies
are completely cut off.
 Because of appreciable amount undergoing enterohepatic circulation,
cobalimin deficiency develop more rapidly in individuals who
malabsorb B12 then it does in vegans, in whom reabsorption of biliary
cobalamin is intact.

6. Components and mechanism for cobalamin absorption

7. Cellular uptake and intracellular reactions involving cobalamin

8. Sufficiently severe to cause
megaloblastic anemia
Malaabsorption of B12, not
usually sufficiently severe &
prolonged to cause anemia
Causes of cobalamin deficiency

9. Pernicious anemia of adults
 Gastric causes of Cobalamin malabsorption
 Severe lack of intrinsic factor due to gastric atophy.
 Man and women ratio is 1:1.6 .
 Peak age of onset is 60 years and with only 10% patients being < 40
years, blacks indivisuals and latin americans the age of onset generally
is lower.
 Common in north europens but occur in all countries and ethnic
groups.
 The disease occur more commenly than by chance in close relatives
and in persons with other orgenic specific autoimmune diseases, e.g.
Thyroid disease, vitiligo, hypoparathyrodism and addison’s disease.
 It also associated with hypogammaglobulinemia, with premature
graying or blue eyes and person of blood group A.
 The serum gastrin level is raised and serum pepsinogen I level are low.

10.  This usually show atrophy of all layers of the body and fundus with
loss of glandular elements, an absence of parietal and chief cells
and replacement of mucous cells, a mixed inflammatory cell
infiltrate and prehaps intestinal metaplasia. The infiltrate of plasma
cells and lymphocyetes contains an excess of CD4 cells.
Gastric biopsy; histological features of stomach in pernicious anemia compared to
normal

11. Juvenile pernicious anemia
 This occurs in old children and resembles PA of adults.
 Gastric atrophy, achlorhydria and serum IF antibofies are all present,
although parietal cell antibodies are usually absent,
 About of patients show an associated endocrinopathy such as
autoimmune thyroiditis, addison’s disease, or hypoparathyroidism; in
some, mucocutaneous candidiasis occurs.

12. Folate
 Folic acid is parent compound of large family of natural folate
compounds.
 Its highest concentration is found in liver, yeast, spinach, other
greens and nuts.
 Folate easily destroyed by heating, particularly in large volumes of
water.
 Total folate in adult is ~10mg with liver containing the largest store.
 Daily adult requirements are ~10ug and so stores are sufficient for 3-
4 months in normal adult and severe folate deficiency may develop
rapidly.

14. Folates absorption in small intestine
& transport
Folate
polyglutamates
Monoglutamates
Lumen-mucosa
5-methyl THF
Intestinal-mucosa
Albumin bound
1/3
unbound 2/3 in
plasma
Cellular
receptors;
PCFT/HCPI,
Clathrin-coated
pits
Membrane
folate
Transporter
Cell cytoplasm
Plasma,CSF, milk
and bile have
folate
In malabsorption syndrome; due to loss of folate 60-90 ug in bile each day,
with folate of sloughed intestinal cells, accelerate speed with which folate
deficiency develop in malabsorption conditions.

15. Causes of folate deficiency

16. Biochemical basis of megaloblastic
anemia
 The folate is needed as coenzyme 5,10-methylene THF polyglutame
for conversion of dUMP to dTMP.
 In deficiencies of folate or cobalamin, there is failure to convert of
dUMP to dTMP; precursor of dTTP.
 The availablity of 5,10-MTHF is reduced in either cobalamin or folate
deficiency.
 in cobalamin deficiency due to failure of formation of THF, the
substrate on which folate polyglutame are build, MTHF accumulates
in plasma and intracsellular concentration fall; termed as THF
starvation or methylfolate trap.

17. The role of folates in DNA synthesis and formation of SAM which is involved in
methylation reaction

18. Bridge to pharmacology
 A substance inhibiting cell growth by competing with, or substituting
for, a natural substrate in an enzymatic process is called
antimetabolities.
 Antimetabolite drugs that interfere with either DNA replication or
folate metabolism may interfere in replicationa and cause
megaloblastic appearance.
 The drugs Methotrexate, hydroxyurea, cytosine arabinoside,
Pyrimethamine and sulfonamides (mainly in bacteria Trimethoprim)
inhibits DHF reductase and so prevent formation of active THF
coenzyme from DHF.

19. Pathogensis
 Megaloblastic anemia are group of disorders characterized by
distinct morphological pattern in hematpoitic stem cells. A
common feature is a defect in DNA synthesis with lesser alterations in
RNA and protien synthesis, leading to state of unbalanced cell
growth and impaired cell division. Most RBCs are not resting but
vainly engaged in attempting to double their DNA,with frequent
arrest in S phase and lesser degree of arrest in other phases of cycle.
An increased percentage of RBCs have increased DNA content is
expressed morphologically as larger than normal “immature nucli”
with finely particulate chromatin whereas the relatively unimpaired
RNA and protein synthesis results in larger cells with greater “mature”
cytoplasm and cell volume. The microscopic appearance of this
nuclear-cytoplasmic asynchrony is morphologically discribed as
“megaloblastic”.

21. Clinical features
 Many patients are diagnosed through finding of a raised MCV on
rotuine CBC.
 The main clinical features in more severe cases are those of anemia.
 Anorexia is usually marked and there may be weight loss, diarrhea or
constipation.
 Glossitis, angular cheilosis and mild fever in more severely anemic
patients, Juandice(unconjugated) and reversible skin melanin
hyperpigmentation also may occur with deficiency of cobalamin or
folates.
 Thrombocytopenia sometimes leads to bruising and this may be
aggrevated by vitamin C deficiency or alcohol in malnourished
patients.
 Anemia and low leukocyte count may predispose to infections,
particularly of respiratory and urinary tract.
 Cobalamin deficiency has also been associated with impaired
bactericidal function of phagocytes.

24. Diagnosis of megaloblastic anemia
 Hematological findings
1. Peripheral blood
2. Bone marrow
3. Ineffective hematopoiesis
4. Chromosome abnomalities
 Diagnosis of cobalamin and folate deficiencies
1. Serum cobalamin
2. Serum folate
3. Red cell folate
4. Serum methylmalonate and homocysteine

25. How to classify the patient of
anemia?

29. Automated enzyme-linked
immunosorbent assay (ELISA)
 Serum cobalamin (normal range; 118-148 pmol/L, MA <74 pmol/L) is
cost effective and most convient to rule out B12 deficiency in vast
majority of patients suspected of having this problem.
 Serum folate (normal range; 11-82 nmol/L; also recent recent diet)
concentation rises in severe B12 deficinecy because of block in
conversion of MTHF to THF inside cells; raised levels has also been
reported in intestinal stagnant loop syndrome due to absorption of
bacterially synthesized folate.

31. Red cells folate assay
 Normal range 880-3520 umol/L.
 Less effected than serum assay for recent diet and traces of
hemolysis.
 False normal results may occur if a folate-deficient patient may
received blood transfusion or a patient has raised reticulocyte
count.

32. Serum methylmalonate and
homocysteine
 For early diagnosis of B12 deficiency even the absence of
hematological abnormalities or subnormal levels of serum
cobalamin can be measured by MMA and homocysteine.
 Serum homocysteine may be raised in other conditions like chronic
renal disease, alcoholism, smoking, pyridoxine deficiency,
hypothyroidism and therapy with steroids, cyclosporine and other
drugs.

33. Ineffective hematopoiesis
 Accumulation of unconjugated bilirubin in plasma , raised urine
urobilinogen, reduced heptaglobin and positive urine hemosiderin,
and a raised lactate dehydrogenase due to death of nucleated
RBCs in marrow.
 A weakly positive direct antiglobulin test due to complement can
lead to false diagnose of autoimmune anemia.

34. Chromosome abnormalities
 Bone marrow cells, transformed lymphocytes, and other
proliferating cells in the body show a variety of changes, including
random breaks, reduced contraction, spreading of centromere and
exaggeration of secondary chromosomal contractions and over-
prominent satellites.

35. Treatment of megaloblastic
anemia
 For out-door patients; establish which of two deficiencies, folate or
B12 is the cause and treat only with appropriate vitamin.
 For In-door patients; once blood sample and bone marrow biopsy
(only if deemed necessary) has been taken for assays, it may be
necessary to treat with both vitamins in large doses.

36. Indications of therapy
 A well-documentated megaloblastic anemia or other
hematological abnormalities and neuropathy due to B12
deficiency.
 Partial or complete gastroectomy or ileal resection and long-term
PPI therapy should be screened and if necessary given B12 therapy.
 As anemia of B12 deficiency responds to folic acid in large doses
before large doses of folic acid are given, Cobalamin deficiency
must be excluded, and if present, corrected and otherwise
cobalamin neuropathy may develop despite a response of anemia
of cobalamin deficiency to folate therapy.

37. Indications for treatment
Cobalamin(B12)
 Replenishment therapy 1000-ug IM
injection of hydroxycobalamin
given at 3-7 days intervals
 Maintenance therapy; 1000 ug IM
every 3 months or 1000-2000 ug
VO daily
 Allergic reactions are rare and
require desensitization or
antihistamine or glucocorticoid
cover.
Folic acid(B9)
 Replenishment therapy; 5-15 mg
daily continue usually till 04 months
when all folate-deficient red cells
will have been eliminated and
replaced by new folate replete
popultation.
 Maintenance therapy; daily use of
food fortification with folic acid

43. Hasta la victoria siempre!

44. Bibliography
 Basic Principles & practice of Hematology 2013 by Hoffman
 Harrison´s principles of internal medicine
 Robbins Basic Pathology
 Guyton & Hall Text of Medical Physiology