Megaloblastic anemia mak

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

2. Differential Diagnosis of Macrocytic Anaemia Megaloblastic Anemia Vitamin B 12 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 B 12 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

5. Absorption and metabolism of vitamin B 12

6. Role of B 12 and Folic acid in DNA synthesis

7. Causes of Cobalamine Deficiency Reduced intake Malabsorption – Addisonian pernicious anemia, Gastrectomy , pancreatic dysfunction, Tropical sprue , Zollinger E llison syndrome Food cobalamine malabsorption - Atrophic gastritis with achlorhydria Abnormal transport protein- Tco I/II deficiency Inborn error of cobalamine metabolism Acquired drug effects

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

9. MORPHOLOGY Certain morphologic features are common to all forms of megaloblastic anemia Bone Marrow 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

10. Bone marrow is hypercellular with cellularity > 95%

11. 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

12. Megaloblasts – Bone Marrow

13. 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)

14. Giant metamyelocytes

15. 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

16. 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

17. 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

18. Diagnosis of Cobalamin Deficiency Established by one of methods Therapeutic trial Serum cobalamin assay Methylmalonic Acid and Homocysteine Assay Deoxyuridine Suppression test Serum holotranscobalamin

19. 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

20. 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

21. 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

22. 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 ( 3 H-Tdr), normal<10% incarporated to DNA An abnormal deoxyuridine suppression test indicates cobalamin or folate deficiency

23. 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

24. 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

25. 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

26. 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

27. 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

28. 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 Anti–parietal cell antibodies 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

29. 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

30. 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

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

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

33. 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

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

35. Differential diagnosis with increased MCV

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