from wintrobe

1. Megaloblastic
anemia
Dr Mohamid Afroz Khan
Moderator-Dr Namrata Shetty

2. Differential Diagnosis of Macrocytic
Anaemia
Megaloblastic Anemia
• Vitamin B12
deficiency
• Folate deficiency
• Drug Induced anemia
Non megaloblastic Anemia
• Excess alcohol consumption
• Liver disease
• Hypothyroidism
• Aplastic anemia
• Paraprotenemia(Myeloma)
• Pregnancy
• Neonatal period

3. Megaloblastic Anemia
• Megaloblastic anemias are associated with
defective DNA synthesis and therefore, abnormal
RBC maturation in the bone marrow (a nuclear
maturation defect)
• However, the primary defect in DNA replication is
usually due to depletion of thymidine triphosphate
which leads to retarded mitosis, and therefore
retarded nuclear maturation
• The depletion of thymidine triphosphate is usually
due to a deficiency of vitamin B12 or folic acid

4. • RNA synthesis is less impeded than is DNA synthesis
hence cytoplasmic maturation and growth continues
accounting for enlargement of the cells
• Increase in total erythropoiesis that may be up to
three times normal
• Decreased rate of appearance of iron in the Hb of
circulating erythrocytes and reticulocytopenia
indicate ineffective erythropoiesis
• Increased destruction of defective erythroid
precursors in the marrow, survival of circulating
erythrocytes is short, indicating hemolysis

6. Absorption and metabolism of vitamin B12

7. Role of B12 and Folic acid in DNA synthesis

8. Causes of Cobalamine Deficiency
1. Reduced intake
2. Malabsorption – Addisonian pernicious anemia,
Gastrectomy, pancreatic dysfunction, Tropical
sprue, Zollinger Ellison syndrome
3. Food cobalamine malabsorption- Atrophic gastritis
with achlorhydria
4. Abnormal transport protein- Tco I/II deficiency
5. Inborn error of cobalamine metabolism
6. Acquired drug effects

9. Causes of Folate Deficiency
1. Reduced Intake
2. Malabsorption- coeliac disease, tropical spure,
small bowel resection, malabsorption syndrome
3. Drug Effect- Sulfa drugs, MTX, OCP, anticonvulsants
4. Increased folate turnover- pregnancy progressive
fall, breastfeeding, skin disease(psoriasis &
exfoliation), haemodylasis, PNH,
haemoglobinopathy, autoimmune haemolytic
anemia

10. MORPHOLOGY
• Certain morphologic features are common to all forms
of megaloblastic anemia
Bone Marrow
1. The bone marrow is markedly hypercellular and
contains numerous megaloblastic erythroid
progenitors
• Megaloblasts are larger than normal erythroid
progenitors (normoblasts) and have delicate, finely
reticulated nuclear chromatin (indicative of nuclear
immaturity)
• As megaloblasts differentiate and acquire Hb, the
nucleus retains its finely distributed chromatin and
fails to undergo the chromatin clumping typical of
normoblasts

11. Bone marrow is hypercellular with cellularity > 95%

12. Comparison of normoblasts (left) and megaloblasts
(right)—B M aspirate
Megaloblasts are larger, have relatively immature nuclei
with finely reticulated chromatin, and abundant basophilic
cytoplasm

13. Megaloblasts – Bone Marrow

14. 2. The granulocytic precursors also demonstrate NC
asynchrony, yielding giant metamyelocytes
• The giant metamyelocyte is the most characteristic
of the abnormal granulocyte
3. Megakaryocytes may also be abnormally large
and have bizarre multilobed nuclei.
• Automated parameter on cell counters the mean
platelet volume is decreased and there is increased
platelet anisocytosis, as detected by the platelet
distribution width (PDW)

15. Giant metamyelocytes

16. Periphral Blood Smears
• Macrocytic anemias associated with magaloblastosis
differs from nonmegaloblastic macrocytic anemia in
that macro-ovalocytes and giant hypersegmented
neutrophils in blood
• Pancytopenia is the rule
• Anemia is macrocytic with an elevated MCV and
extreme degree of anisocytosis and poikilocytosis
• Microcytes are common

17. • Basophilic stippling, multiple Howell-Jolly bodies,
nucleated red cells with karyorrhexis and cabots ring
may be seen
• Leucopenia is present
• Thrombocytopenia usually seen, rarely sufficiently
severe to be responsible for bleeding
• Neurologic symptoms may be present in the absence
of anemia
• Reticulocytopenia, increased S.Fe and elevated S.LDH

19. Hypersegmented neutrophils
• Earliest change in periphral blood even before
devlopment of anemia
• Increased lobe size as well as number of nuclear
segments
• Neutrophil hypersegmentation can be defined as
the presence of neutrophils with six or more lobes
or the presence of more than >3% of neutrophils
with at least five lobes

21. Diagnosis of Cobalamin Deficiency
• Established by one of methods
1. Therapeutic trial
2. Serum cobalamin assay
3. Methylmalonic Acid and Homocysteine Assay
4. Deoxyuridine Suppression test
5. Serum holotranscobalamin

22. Therapeutic trial
• With the patient on a diet low in cobalamin and
folate
• Parenteral physiologic dose of cobalamin (10
µg/day) is given
• Optimal hematologic response indicates deficiency
and consists of reticulocytosis beginning on the
third or fourth day, reaching a peak on the
seventh day
• Erythropoiesis becomes normoblastic by 2 days, and
leukopoiesis becomes normal by 12 to 14 days.
• Within a week, leukocyte and platelet counts have
returned to normal, and the Hb concentration begins
to rise

23. Serum Cobalamin Assay
• Reference values are 200–900 ng/L
• In megaloblastic anemia due to cobalamin
deficiency, serum cobalamin is usually less than
100 ng/L
• Individuals with folate deficiency and mild cobalamin
deficiency have borderline values between 100 and
200 ng/L as in pregnancy
 Microbiological assay (Euglena gracilis),
 Radioisotopic dilution
 chemiluminescence assays

24. Methylmalonic Acid and Homocysteine Assays
• Cobalamin coenzyme is essential for the
isomerization of methylmalonate to succinate
• Urine excretion of increased amounts of
methylmalonate is found in cobalamin deficiency
• Plasma levels of methylmalonic acid and
homocysteine are increased
• Interpreted with caution in patients with chronic
renal failure

25. Deoxyuridine Suppression Test
• Measures the ability of marrow cells in vitro to
utilize deoxyuridine in DNA synthesis
• Normally, in marrow cells, the major source of
thymidine for DNA is de novo synthesis from
deoxyuridine, which requires intact cobalamin and
folate enzymes
• Tritium-labeled thymidine (3H-Tdr), normal<10%
incarporated to DNA
• An abnormal deoxyuridine suppression test indicates
cobalamin or folate deficiency

26. Serum holotranscobalamin
• Early marker of B12 deficiency
• Low levels <23mmol/l often as low as 5mmol/l in
pernicious anaemia
• Subject to recent dietary change, within 24 h
• Particularly useful in pregnancy, where levels
unaffected by trimester

27. Detecting the Cause of Cobalamin Deficiency
• Clinical history is useful in suggesting whether
cobalamin or folate deficiency is the cause of
megaloblastic anemia
• Clinical associations of pernicious anemia include a
family history of PA in one third of patients
• certain endocrine deficiencies -thyroid disease,
diabetes mellitus, hypothyroidism, and Addison’s
disease
• Immune disorders (immune thrombocytopenic
purpura, autoimmune hemolytic anemia, and
acquired hypogammaglobulinemia

28. Diagnosis of Folate Deficiency
Serum and Red Cell Folate
• Microbiological assay employing Lactobacillus casei
is reliable method for definitive diagnosis
• Radioisotopic and chemiluminescence methods
widely used due to rapidity and greater
convenience
• In cobalamin deficiency, serum folate is decreased
in 10% of cases, increased in 20%, and normal in the
remainder

29. • Unlike serum folate (entirely 5-methyltetrahydrofolate),
red cell folates are a heterogeneous mixture of
different forms with varying polyglutamate chain
lengths
• The red cell folate is a better test of body folate stores
and is decreased in megaloblastic anemia due to folate
deficiency
Urinary Formiminoglutamic Acid
• Useful in megaloblastic anemia due to antifolate drugs

31. Plasma Homocysteine Assay
• As with cobalamin deficiency, total plasma
homocysteine is increased in approximately 75%
of patients with folate deficiency
• The level of methylmalonic acid is normal

32. Pernicious Anemia
• Most common cause of cobalamin deficiency
• caused by failure of the gastric mucosa to secrete
intrinsic factor
• Abnormality is genetically determined & manifested late
in life >40 years
Immune Abnormalities
1. Anti–parietal cell antibodies
2. Anti–intrinsic factor antibodies -Two types
• Blocking” antibodies, which block the binding of
cobalamin to IF
• Binding” antibodies, which bind to the cobalamin–IF
complex and prevent the complex from binding to
receptors in the ileum

35. Schilling Test
• Determine whether IF is lacking
• Measures radioactivity in a 24-hour sample of urine
• Two hours after oral administration of 0.5–2.0 µg
of radioactive cobalamin, a large “flushing” dose
of nonlabeled cobalamin is given parenterally
• Normal individuals will excrete more than 7% of a 1-
µg dose of ingested cobalamin in the urine in 24
hours
• Patients lacking IF will excrete less

37. • If excretion is low, the test must be repeated using the
same procedure, except that hog IF is given orally,
along with labeled cobalamin
• If 24-hour excretion is normal, the low value in the
first part was due to IF deficiency
• If excretion remains abnormal in the second part of
the procedure, an explanation for malabsorption of
cobalamin on the basis of intestinal disease must
be sought.
• The test may be repeated after 7–10 days of
antibiotic administration if bacterial overgrowth is
suspected, and pancreatic extracts may be added to
investigate the possibility of pancreatic dysfunction

38. Morphology
• Changes in BM and Blood are similar to all
megaloblastic anemia
• In addition it leads to
1. Atrophic glossitis
2. Intestinalization of gastric mucosa
3. In CNS principal alteration in spinal cord causing
degeneration of myelin in dorsal and lateral tract

39. Non-megaloblastic macrocytic anemia
• The macrocytic RBCs are not oval, but are
round.
• There are no hypersegmented neutrophils or
Howell-Jolly bodies

41. Liver Disease
• Liver disease associated with alcoholism may
lead to folate-deficient megaloblastic anemia
• Because of the grossly inadequate diet of the
alcoholic, and because the liver is the major
site for folate storage and metabolism
• With adequate dietary folic acid intake, however,
the anemia that is found with liver disease is
macrocytic and normoblastic—not megaloblastic

42. • Red cells fairly uniform
in size and shape
• Macrocytosis
• Stomatocytosis
• Derranged LFT

43. Differential diagnosis with increased MCV

45. Thank
You