Anemia:- Part 3 – Megaloblastic Anemias, Macrocytic, Vitamin B12 and Folic Acid Deficiency, and Lab findings

Sample

1. Prepare the peripheral blood smears.
   1. Can take blood in the EDTA.
   2. Also, make a direct fresh blood smear.
2. Take blood for the study of Vit. B12 and folic acid.
3. Bone marrow examination may be needed.

Pathophysiology

1. Megaloblastic anemia is a subgroup of macrocytic anemia which is characterized by defective nuclear maturation. There is defective deoxyribonucleic acid (DNA) synthesis.
2. Vitamin B12 and folic acid are needed for the synthesis of thymidine triphosphate and convert Homocysteine to Methionine.
   

   Megaloblastic anemia mechanism

2. There are megaloblasts, large and abnormal RBCs precursors in the bone marrow, and macrocytes in the peripheral blood smear.
   

   Megaloblastic anemia red blood cell immature in the bone marrow

3. White blood cells show giant metamyelocytes in the bone marrow are a characteristic feature.
   

   Megaloblastic anemia giant metamyelocyte

4. Megakaryocytes are also abnormal.

5. Megaloblastic anemia may be of three types:
   1. Anemia is caused by folate deficiency.
   2. Anemia caused by Vit.B12 deficiency.
   3. Anemia where there is no response to either of the treatment. This third group does not respond to therapy by vitamin B12 or folic acid.
6. Vitamin B12 deficiency causes and mechanism:
   1. Vitamin B12 sources are meat, eggs, and dairy products.
      1. Gastric parietal cells secrete the Intrinsic factor, which binds to B12 and allows its absorption in the ileum.
         

         Parietal cells of the stomach secrete the Intrinsic factor.

         

         Megaloblastic anemia mechanism

         

         Absorption of the vitamin B12 mechanism

   2. Deficiency of vitamin B12 causes are:
      1. Decreased intake in the diet.
      2. Poor diet.
      3. For vegetarians.
      4. Malabsorption due to any cause, e.g., gastrectomy (partial or complete), antibodies to intrinsic factor, sprue, celiac disease, malignancies (lymphomas),  and parasitic infestation.
   3. Intestinal causes:
      1. Jejunal diverticulosis.
      2. Blind loop stricture.
      3. Ileal resection.
      4. Fish tapeworms.
   4. Increased demand of the body:
      1. During pregnancy.
      2. Hyperthyroidism.
      3. Malignant tumors.
   5. Enzyme deficiency.
7. Folate Deficiency is seen in:
   1.  Decreased intake.
      1. Poor diet.
      2. Inadequate use of vegetables.
      3. In alcoholism.
   2. Malabsorption like:
      1. Steatorrhea.
      2. Tropical sprue.
      3. Celiac disease.
      4. Gluten-induced enteropathy.
      5. Partial gastrectomy (some cases).
      6. Extensive jejunal resection.
   3. Pathological causes like:
      1. Hemolytic anemia.
      2. Myelofibrosis.
      3. Malignant diseases like:
         1. carcinoma.
         2. Myeloma.
         3. Lymphoma.
   4. Increased demand of the body:
      1. Infancy.
      2. Pregnancy.
      3. Hyperthyroidism.
      4. Malignant tumors.
   5. Drugs:
      1. Phenytoin.
      2. Oral contraceptives.
   6. Inflammatory diseases of GIT like:
      1. Crohn’s disease.
      2. Tuberculosis.
      3. Psoriasis.
      4. Rheumatoid arthritis.
      5. Exfoliative dermatitis.
   7. Drugs like:
      1. Folic acid antagonist likes triamterene and trimethoprim.
      2. Anticonvulsant.
      3. Sulfasalazine.
   8. If there is an enzyme deficiency.
      

      Differential diagnosis of megaloblastic anemia

Clinical presentation

1. The onset is insidious, and gradually signs and symptoms of anemia appear.
2. The patient may have mild jaundice due to an excessive breakdown of RBCs.
3. There is glossitis, which is the beefy- red sore tongue.
   1. There is angular stomatitis.
4. The patient may have weight loss due to malabsorption.
5. There is purpura due to thrombocytopenia.
6. There is pigmentation; the cause is unknown and may be the presenting feature.
7. In the case of Vitamin B12 or Folate, deficiency leads to:
   1. Megaloblastic anemia.
   2. Neuropathy is only due to a deficiency of Vitamin B12.
   3. Melanin skin pigmentation.
   4. Infertility.
   5. There is a decreased osteoblastic activity.
      1. There may be a neural tube defect in the fetus.

Laboratory findings are:

• MCV is increased to 110 to 115 fl, where the normal value is  77 to 93 fl.
• MCH is slightly increased where the normal value is 27 to 32 pg.
• MCHC is within normal limits where the normal value is 20 to 25 g/dl.
  
  1. Low hemoglobin. Hemoglobin is typically low (may reach 2 g/dL).
  2. MCV > 99 fL, Increased MCV (>95 ) may reach 100 to 140 µm².
  3. The peripheral blood smear shows macrocytosis and many hypersegmentation of neutrophils.
  4. Occasionally may see leucopenia and thrombocytopenia.
  5. Reticulocytes are not in comparison to the degree of anemia.
     

     Differential diagnosis of macrocytic anemia

Peripheral blood picture:

1. Hb is reduced to 9 to 10 G /dl.
2. The total RBC count is reduced.
3. WBCs are normal or decreased. There is around 5% Poly with more than 5 lobes, and normally these have 3 to 4 lobes.
4. DLC shows marked neutropenia and relative lymphocytosis. There are macrocytes (macroovalocytes) or hypersegmented neutrophils; these are characteristics of megaloblastic anemia.
5. Peripheral blood smears show anisocytosis, poikilocytosis with Macrocytes’ presence (MCV = 100 to 140 µm²).

5. Myelocytes may be seen.
6. RBCs show  Macrocytosis, anisocytosis, poikilocytosis, polychromasia, punctate basophilia, and occasionally nucleated RBCs.
7. Platelets show thrombocytopenia and with the presence of giant platelets.
8. Reticulocytes are normal or decreased.
9. Reticulocyte index is used to evaluate effective RBCs production.
   1. Normally increased after blood loss or hemolysis.
   2. Decreased in:
      1. Nutritional deficiency.
      2. Marrow aplasia or replacement.
      3. Exposure to toxic agents.
10. Biochemical findings are:
    1. High serum iron.
    2. Indirect bilirubin is high.
    3. Serum LDH is raised.
    4. When there is a high gastrin level indicates pernicious anemia.
    5. Vitamin B12 level is low in B12 deficiency in serum and RBCs.
    6. The folic acid level is low in folic acid deficiency in serum and RBCs.
    7. Antibodies to gastric cells and intrinsic factors are seen in pernicious anemia.
    8. Abnormal Schilling test is seen in pernicious anemia.

Bone marrow:

1. Normally it is not needed to make the diagnosis of vitamin B12 deficiency.
2. It is hyperplastic with megaloblastic erythropoiesis.
3. Megaloblasts and normoblasts are seen.
4. Megaloblastic changes with the degree of severity of the anemia.
5. Leucopoiesis is abnormal. Macropolycytes are seen. There are hypersegmented nuclei.
6. The myeloid and erythroid ratio is reduced.
7. Megakaryocytes are usually normal or slightly increased.
8. Hemosiderin storage is normal.
9. Vitamin B12 serum level is reduced where the normal values are 160 to 925  ng/dl.
   1. Vitamin B12 level in the RBCs is also decreased where the normal value is 72 to 512 ng/dl.
10. Serum Folate level is also decreased in the folic acid deficiency, where the normal value is 10 to 15  µg /L.
    1. RBC folate is also reduced and may be very low. The normal value is 300 to 350 µg /L.
11. Iron in plasma is raised or normal.
12. Serum bilirubin is moderately raised and is predominantly unconjugated bilirubin.
13. Serum LDH is also raised due to ineffective erythropoiesis. In severe cases, there is a markedly increased level.

Diagnostic tests are:

1. 1. The diagnostic test is an assay of Vit B12, which is <90 pg/mL.
      1. The associated normal level of folate indicates a B12 deficiency.
   2. The diagnostic test for Folate is the estimation of the folate level.
      1. If folate is <3 ng/mL with a normal B12 level suggests Folate deficiency.
   3. Serum homocysteine level increases with B12 or folate deficiency.
   4. Antibodies against intrinsic factors strongly suggest B12 deficiency (pernicious anemia).
   5. Urine methylmalonic acid levels are increased in B12 deficiency.
   6. Schilling test is also diagnostic.

Megaloblastic anemia  features and differential diagnosis:

Clinical characteristics	Serum vitamin B12 deficiency	Serum folate deficiency	RBC folate level
Diet	Rare and seen in strict vegetarians	Commonly seen in alcoholics.
Etiology for malabsorption	Post gastrectomy Pernicious anemia Pancreas insufficiency Diphylobothum latum infestation Crohn’s disease	Sprue
Increased demand for the body	Increased RBCs destruction Pregnancy	Pregnancy Increased RBCs destruction
Effect of drugs	Dilantin sodium	Methotrexate
Inheritance	Transcobalamine II deficiency	None
Normal biochemical picture	200 to 900 pg/mL	5 to 16 ng/mL	>150 ng/mL
Vit. B12 deficiency	Low <100 pg/ml	Normal or high (>16 ng/mL)	Low  <150 ng/mL
Folic acid deficiency	Normal 200 to 900 pg/mL	Low < 3 ng/mL	Low (better test)
Deficiency of both	Low	Low	Low

Summary of the Vitamin B12 and Folates deficiency and lab findings:

Parameters	Results (Lab findings)
Peripheral blood smear and complete blood count	Anisopoikilocytosis is variable Hypersegmented neutrophils Oval macrocytes Pancytopenia in severe cases
Bone marrow	Hypercellular marrow Immature RBC Giant metamyelocytes. Megaloblastic marrow Erythroid hyperplasia with the shift to left
Vitamin B12 deficiency shows:	Increased indirect bilirubin Increased LDH Decreased serum B 12 level Decreased RBC folates Increased urine methylmalonic acid
Folate (folic acid) deficiency shows:	Increased indirect bilirubin Increased LDH Decreased RBC folates Decreased serum folates Increased urinary formiminoglutamic acid (FIGLU)

Clinical and laboratory findings in Vitamin B12 and Folate deficiencies:

Lab/clinical parameters	Pernicious anemia (Vit.B12 Deficiency)	Folate deficiency
MCV	Increased	Increased
Hypersegmented neutrophils	Present	Present
Pancytopenia	Present	Present
Serum gastrin level	Increased	Increased
Urine methylmalonic acid	Increased	Normal
Plasma homocysteine	Increased	Increased
Autoantibodies	Present	Absent
Chronic atrophic gastritis	Present	Absent
Risk for gastric carcinoma	Present	Absent
Achlorhydria	Present	Absent
Neurologic disease	Present	Absent

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