1. MEGALOBLASTIC ANEMIAS
Tabuk University
Faculty of Applied Medical Sciences
Department Of Medical Lab. Technology
2nd Year – Level 4 – AY
Hematology – 1, MLT 205
MEGALOBLASTIC ANEMIAS
Mr. Waggas Ela’as, M.Sc, MLT

2. Objectives
Define Macrocytic anemias and relate them to megaloblastic anemias.
Discuss the normal metabolism of B12 & folic acid
Discuss the role of B12 & folic acid in nuclei maturation
Describe the clinical presentation of megaloblastic anemias.
List and discuss lab findings and diagnosis of megaloblastic anemias.
Compare megaloblastic anemias caused by B12 deficiency with folic acid deficiency.
Discuss and describe Pernicious anemias and Schilling test.

3. MACROCYTIC ANEMIAS
These are the anemias in which the RBC have an MCV of greater than 96 fl.
There are 2 groups of macrocytosis
Megaloblastic anemia
Non megaloblastic macrocytosis, which includes :
Alcohol, Liver disorders, cyotoxic drugs, a plastic anemia, pregnancy, smoking, reticulocytosis, neonatal, myeloma, Hypothyroidism , Myelodysplastic syndrome , Scurvy (Vit-C dif)

4. MEGALOBLASTIC ANEMIAS
Group of disorders in which erythroblasts in the bone marrow, show delayed nuclear maturation relative to that of cytoplasm, due to defective DNA synthesis. When DNA synthesis is impaired, the cell cycle cannot progress to mitosis. This leads to continuing cell growth without division, which presents as macrocytosis.

5. Causes of Megaloblastic anemia
The most common causes are :
1. B12 deficiency
2. Folic acid deficiency.
Other causes include :
Abnormalities of B12 or folate metabolism (e.g : transcobalamin deficiency, nitrous oxide, anti folate drugs)
Defects in DNA synthesis ( congenital or acquired enzyme deficiency)

6. B12 (cobalamin)
Synthesized ONLY by microorganisms, and animals acquire it by production from intestinal bacteria, by eating other animal foods or by eating bacterially contaminated foods.
Found in liver, meat, fish, dairy products.
Does not found in fruit, cereals or vegetables.

7. Absorption of B12
In stomach, Bl2 is combined with the intrinsic factor (IF) which is synthesized by the gastric parietal cells.
The IF-B12 complex can then bind to a specific surface receptor in distal ileum where it is absorbed and IF destroyed.
Vitamin Bl2 is absorbed into portal blood where it becomes attached in the plasma to transcobalamin (TC) which delivers Bl2 to bone marrow and other tissues.
(Note the similarity between Transcobalamin & Transferrin)

8. Megalobastic anemia due to B12 deficiency, occurs in TC deficiency, although serum B12 is normal. Why?
Because B12 is bound to Haptocorrin (an other plasma protien synthesized by granulocytes & macrophages).
Haptocorrin Is unable to deliver B12 to bone marrow and other body cells.
Function of B12 : needed in the conversion of Methyl THF to THF, a step in the DNA synthesis.

9. Causes of Vit.B12 deficiency
Nutritional - low intake : vegans
Malabsorption
Gastric causes (Pernicious anemia, congenital lack of IF, total or partial gastrectomy).
Intestinal causes (tropical sprue)
Fish tapeworm. (Diphyllobothrium latum absorbing 80% or more of the host’s B12 intake)
(Tropical sprue is a chronic diarrheal disease, possibly of infectious origin, that involves the small intestine and is characterized by malabsorption of nutrients, especially folic acid and vitamin B12)

10. Folic acid Is a parent for a large group of compounds, the folates.
Humans are unable to synthesize folate structures.
Leafy vegetables are a principal source
Folate is needed in many biochemical reactions in DNA synthesis. Folate as 5,10 methylene tetrahydrofolate (THF) polyglutamate, is required in the synthesis of thymidine monophosphate to form eventually the DNA and to repair it.
It has no specific plasma protien facilitate transport.

11. Causes of Folic acid deficiency
1. Nutritional (old age, poverty, special diets)
2. Malabsorption :
Tropical sprue, partial gastrectomy, jejunal resection)
3. Increased requirements:
- pregnancy and lactation
- infancy
- malignancy
- hemodialysis
4. Excess urinary loss
(active liver disease, congestive heart failure)
5. Drugs (anticonvulsants)
5. Mixed (liver disease, alcoholism)

12. VITAMIN B12 AND FOLIC ACID- nutritional aspect
Liver, greens & yeast
Animal produce only
Main foods µg
1 - 2 µg
Minimal Daily requirement
10-12 mg (sufficient for 4 months)
2 – 3 mg (sufficient for 2-4 years)
Body stores
Easily destroyed
Little effect
Cooking
duodenum & jejunum
ileum
Places of absorption
Conversion to MTH
Intrinsic factor
Absorption
3.0 – 15.0 µg/l
160 – 925 ng/l
Normal values in serum

13. MEGALOBLASTIC ANEMIAS clinical features
1. Symptoms of anemia
2. Symptoms associated with vit. B12 or Folic acid deficiency
◙ Neurologic symptoms (exclusivly in severe Vitamin B12 deficiency) : numbness خدر and tingling تنميل in the hands and feet.
◙ Gastrointestinal (vit.B12 and folic acid deficiency)
- loss of appetite
- glossitis (beefy red, painful tongue)
- angular stomatitis
3. Jaundice due to ineffective erythropoesis.

17. Laboratory Diagnosis
1. Diagnosis of megaloblastic anemia (by doing blood count, blood films, Biochemical tests & bone marrow examination). 2. Establishing a type of deficiency (vit. B12 and/or folic acid) . By estimating serum B12, serum folate, Red cell folate. 3. Establishing a cause of deficiency (by doing absorption tests : e. g : Schilling test)

18. macrocytic anemia ( Low Hb with MCV>100fl )
Diagnosis(1)
A. Blood cell count:
macrocytic anemia ( Low Hb with MCV>100fl )
Low Platelets & Leucocytes count.
low reticulocyte count
B. Blood film :
Macrocytosis (oval macrocytes) , anisocytosis.
hypersegmentation of Neutrophils. (more than 5 lobes nucleus)

19. Hyper segmented neutrophil
Blood film
Hyper segmented neutrophil
Normal Neutrophils

20. Compare the red cell size to the nucleus of small Lymphocytes
Blood film (cont.)
Macrocytic red cells
Normocytic red cells
Compare the red cell size to the nucleus of small Lymphocytes

21. Elevation of lactate dehrogenase (LDH) D. Bone marrow smear
Diagnosis(1)
C. Laboratory findings
Hyperbilirubinemia
Elevation of lactate dehrogenase (LDH)
D. Bone marrow smear
Hypercellular
Increased erythroid /myeloid ratio
Erythroid cell changes (megaloblasts: an abnormally large cell with nuclear- cytoplasmic asynchrony : open, fine, primitive chromatin but normal Hemoglobinization)
Myeloid cell changes : Giant metamyelocytes.
Megakaryocytes are decreased and show abnormal morphology

22. Erythroblasts in bone marrow with open stippled primitive chromatin appearance

23. Diagnosis (2): Establishing the type of deficiency
In Folic acid deficiency
In B12 deficiency
Serum B12 : Normal
Serum B12 : reduced
Serum folate : reduced
Serum folate : increased
Red cell folate : reduced
Without B12 deficiency, red cell folate is more accurate than serum folate for tissue folate status . Why? Because red cells acquire folate when they are produced, and the cellular concentration of folate does not change during the red cell lifespan. A single folate-rich meal can correct the serum folate in a deficient patient, while a persistently low red cell folate reflects chronically depleted stores.

24. Diagnosis (3): Establishing a cause of deficiency (Schilling test)
The Schilling test is used to determine whether the body absorbs vitamin B12 normally.(to find the cause of low vitamin B12 levels).
One µg of radioactive B12 is taken orally.
One hour later, a 1000 µg injection "flushing" dose of non-radioactive B12 is given to saturate B12 binders (Transcobalamines)
Radioactive B12 is excreted in the urine; and assayed for its content of radioactivity.
A second stage may be needed, in which the first step is a combination of B12 and IF.

25. pernicious anemia
Pernicious anemia is an autoimmune disease that affects the gastric mucosa and results in gastric atrophy.
Antibodies are produced against cells of the stomach or against IF.
This leads to the destruction of parietal cells and failure to produce intrinsic factor, resulting in vitamin B12 malabsorption .
If pernicious anemia is left untreated, it causes vitamin B12 deficiency, leading to megaloblastic anemia and neurological disorders, even in the presence of adequate dietary intake of vitamin B12.

26. Homework-4
1. What is the difference between Megaloblastic and pernicious anemia?
2. Which ONE of the following is NOT TRUE about megaloblastic anaemia?
It is always caused by B12 or folate deficiency
Bone marrow appearances are identical in B12 or folate deficiency
It is caused by defective DNA synthesis
It may be caused by nitrous oxide
It is associated with jaundice

27. 3. Case
A 32 year old male is admitted to the emergency department suffering from multiple gunshot wounds to the abdomen. Surgery results in removal of the all of the jejunum. Several months later, the patient complains of being tired. A CBC indicates an increased MCV of 125, decreased Hb and Hct. Which of the following is consistent with these data? Why?
a)iron deficiency anemia
b)anemia of chronic disease
c)Vit B12deficiency
d)Folate deficiency
e)Sideroblastic anemia