Megaloblastic anaemia

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1. MEGALOBLASTIC ANAEMIAS

2. Introduction .. Characterized by defective synthesis of deoxyribonucleic acid (DNA) in all proliferating cells Most commonly result from lack of folic acid or vitamin B 12

3. MEGALOBLASTIC ANAEMIA [ VITAMIN B 12 DEFICIENCY ]

4. Normal Vitamin B 12 M etabolism Vitamin B 12 is composed of A corrin nucleus which has 4 pyrrole rings bound to a central cobalt atom A 5,6 dimethylbenzimidazole group which is attached to the corrin ring and to the central cobalt atom Important cobalamins that are distinguished according to the ligand attached to the central cobalt atom are : cyanocobalamin , hydroxocobalmin , adenosylcobalamin and methylcobalamin

5. Sources Liver, dairy products and seafish are major sources Although bacteria in the large intestine synthesize vitamin B 12 it cannot be absorbed from this site Minimum amount required for an adult is 1 to 4 µg per day

6. Absorption of Vitamin B 12 2 mechanism Active (75%) – requires the presence of intrinsic factor ( a glycoprotein produced by gastric mucosa) Passive – absorption occurs by diffusion and works when pharmacological doses of vitamin B 12 are ingested

7. Vitamin B12 in food R-Binder B12-R-Binder complex IF-B12 complex + Freed R-Binder Intrinsic Factor (IF) Receptor-IF-B12 B12-TCII Circulation Receptor TCII IF Degradation Epithelial cell of terminal iIeum Stomach Duodenum

8. Transport of Vitamin B 12 Following absorption by the ileal mucosal cells, vitamin B 12 is carried in the plasma by various transporting proteins: Transcobalamin I Transcobalamin II Transcobalamin III

9. Transcobalamin I (TC I) is an alpha-globulin produced by granulocytes. It functions as a circulating reserve store of B 12 . TC I carries mostly methylcobalamin . Transcobalamin II (TC II) is a beta-globulin formed in the liver and is the dominant carrier of B 12 immediately after absorption. It is the main agent for rapid transport of B 12 to the body cells. Transcobalamin III (TC III) is an alpha-globulin. TC III may act as a defence mechanism by depriving pathogens of B 12 at sites of infection

10. Storage sites Total amount of vitamin in body is 2-5 mg ( adequate for 3 years ) Major site : liver Excreted through the bile and shedding of intestinal epithelial cells Most of the excreted vitamin B 12 is again absorbed in the intestine ( enterohepatic circulation)

11. Functions of Vitamin B 12 Synthesis of methionine from homocysteine Conversion of methyl malonyl C oA to succinyl CoA

12. FH4 FH2 Methylene FH4 Methyl FH4 Intestinal cell Dietary folates Dihidrofolate Redutase Methionine Homocysteine Thymidylate Synthase DNA Synthesis dTMP dUMP Role of Vitamin B12 and Folate in DNA synthesis VitB12 ( Methylcobalamin )

13. General Morphological Features Of Megaloblastic Anemia

14. PERIPHERAL BLOOD FINDINGS Hemoglobin – decreased Hematocrit – decreased RBC count – decreased/normal MCV - >100fl ( normal 82-98fl) MCH –increased MCHC – NORMAL Reticulocytopenia . Total WBC count – normal / low Platelet count – normal/ low Pancytopenia , especially if anaemia is severe.

15. PERIPHERAL SMEAR RBC: - Macro ovalocytes ( macrocytic normochromic ) [ macrocytosis is the earliest sign in Vit B 12 deficiency and can be detected even before the onset of anaemia ] - In severe anaemia in addition to macrocytosis , marked anisopoikilocytosis , basophilic stippling, howell jolly bodies, Cabot’s rings may be found

16. Late or intermediate erythroblast with fine, open nuclear chromatin ( megaloblast ) may be seen in peripheral blood in severe anaemia

17. Marked macro- ovalocytosis (MCV 134 fl) in the peripheral blood smear of a patient with vitamin B12 deficiency.

18. PERIPHERAL SMEAR WBC Normal count or reduced count Hypersegmented neutrophils is one of the earliest sign of megaloblastic haematopoiesis and can be detected even in the absence of anaemia (when more than 5% of neutrophils show ≥ 5 lobes; 1% neutrophils with ≥ 6 lobes) PLATELETS: Normal or decreased (severe anaemia ) Giant platelet can occur

19. BONE MARROW Markedly hypercellular Myeloid : erythroid ratio decreased or reversed. (Normally, there are three myeloid precursors for each erythroid precursor resulting in a 3:1 ratio , known as the M:E ( myeloid to erythroid ) ratio) Erythropoiesis : MEGALOBLASTIC

20. MEGALOBLAST Cell and nuclear size and amount of cytoplasm (deeply basophilic royal blue) are increased Nuclear chromatin is sieve like or stippled (open) Nuclear cytoplasmic asynchrony/dissociation Abnormally large precursor ( promegaloblast and early megaloblast ) are increased in BM – Maturation arrest Abnormal mitoses (increased)

21. Granulocytic series also display megaloblastic changes Most prominent change – giant metamyelocyte with horseshoe shaped nuclei and finer nuclear chromatin, and in band forms Megakaryocytes are often large with multiple nuclear lobes and paucity of cytoplasmic granules

22. BIOCHEMICAL FINDINGS Increase in serum unconjugated bilirubin - because of ineffective erythropoiesis Increase is LDH Normal serum iron and ferritin

23. Causes of Vit B 12 deficiency Insufficient dietary intake (very rare) Strict vegetarians Deficient absorption Pernicious anaemia Total or partial gastrectomy Prolonged use of PPI or H 2 blockers Diseases of small intestine Fish tapeworm infestation

24. PERNICIOUS ANEMIA Thomas Addison (1849) Disease of elderly – 5 th to 8 th decades (median age at diagnosis – 60 years) Genetic predisposition Tendency to form antibodies against multiple self antigens

25. PATHOGENESIS Immunologically mediated, autoimmune destruction of gastric mucosa CHRONIC ATROPHIC GASTRITIS – marked loss of parietal cells Three types of antibodies: Type I antibody- 75% - blocks vitamin B 12 and IF binding Type II antibody – prevents binding of IF-B 12 complex with ileal receptors Type III antibody – 85-90% patients – against specific structures in the parietal cell

26. Pathological changes are infiltration by mononuclear cells in submucosa and lamina propria of fundus and body of the stomach, progressive loss of parietal and chief cells, and their replacement by intestinal type mucous cells

27. Associated with other autoimmune disorders like Hashimoto’s, Graves’, vitiligo , diabetics mellitus, primary hyperparathyroidism, Addison’s and Myasthenia gravis Patients with pernicious anaemia have increase risk of gastric cancer

28. DIAGNOSTIC FEATURES Moderate to severe megaloblastic anemia Leucopenia with hypersegmented neutrophils Mild to moderate thrombocytopenia Mild jaundice due to ineffective erythropoiesis and peripheral hemolysis Neurologic changes Low levels of serum B 12

29. Elevated levels of homocysteine Striking reticulocytosis after parenteral administration of vitamin B 12 Serum antibodies to intrinsic factor (specific) and anti parietal cell antibodies in serum Abnormal Schilling test, pentagastrin -fast achlorhydria

30. GASTRECTOMY Total gastrectomy : Secondary to Vit B 12 deficiency as it removes the site of synthesis of intrinsic factor Prophylactic vitamin B 12 after surgery Partial gastrectomy Regular follow up after surgery for early detection of deficiency

31. DISEASES OF SMALL INTESTINE Tuberculosis, whipple’s disease, blind loop syndrome or resection of small intestine may interfere with absorption that occurs in the terminal ileum Blind loop syndrome – stasis of small intestine contents ( diverticulum / stricture) may predispose to bacterial colonization and proliferation Utilization of most of the ingested Vit B 12 by bacteria may lead to reduced or non avail of V it for absorption

32. INFESTATION BY FISH TAPEWORM Diphyllobothrium latum (inadequately cooked fish) Vitamin deficiency by competing with the host for vitamin in food Diagnosis made by demonstration of ova in stool

33. CLINICAL FEATURES Anaemia , mild jaundice and sometimes neurological involvement N eurological involvement in the form of Peripheral neuropathy Subacute combined degeneration of spinal cord Cerebral changes (personality changes, dementia & psychosis) Patients can present with only neurological abnormalities without megaloblastic anaemia

34. LABORATORY FEATURES Morpholgical changes of megaloblastic anaemia in PS and BM Serum vitamin B 12 assays Methylmalonic acid (MMA) and homocysteine in serum Schilling test Intrinsic factor antibodies in serum

35. 1. SERUM VITAMIN B 12 ASSAYS Various methods are available, e.g. microbiological methods using Lactobacillus leichmannii or radio-isotope techniques (RIA) using 57 CoB 12 , coated charcoal and IF.

36. RADIO-ISOTOPE DILUTION ASSAY : A known amount of radioactive (hot) B 12 is diluted with the non-radioactive (cold) B 12 in the test serum, released from serum proteins by heat or chemical means. A measured volume of the hot and cold mixture is bound to intrinsic factor (IF) which is added in an amount insufficient to bind all the hot B 12 . The bound B 12 is separated from the free and its radioactivity counted.

37. The count is inversely proportional to the B 12 concentration in the test serum. The higher the serum B 12 the greater will be the dilution of the radioactive B 12 and thus less radioactivity attached to the IF. By comparison with standards of known B 12 content, the B 12 content of the test serum can be calculated.

38. In Vitamin B 12 deficiency , Serum Vitamin B 12 and red cell folate are depressed Serum folate is normal or increased ( accumulation of 5-methyl tetrahydrofolate ) [ folate trap]

39. 2. Methylmalonic acid (MMA) and homocysteine in serum Recent reports of S.methylmalonic acid and S.homocysteine are more sensitive for detection of Vitamin B 12 than estimation of Vitamin B 12 Raised early in tissue deficiency even before appearance of hematological changes

40. 3. SCHILLING TEST For evaluation of absorption of vitamin B 12 in the GIT Performed in 2 parts – part 1 and part 2 Part 1 : 0.5 to 1 µg of radiolabelled vitamin B 12 is given orally After 2 hrs IM dose (1000 µg) of unlabelled vitamin B 12 is given [ saturates binding sites of TC I and TC II and displaces any bound radiolabelled vitamin B 12 (thus permitting urinary excretion of absorbed radiolabelled vitamin B 12 )

41. Radioactivity is measured in subsequently collected 24 hr urine sample and expressed as a % of total oral dose In normal persons, > 7% of the oral dose of vitamin B 12 is excreted in urine If excretion is less than normal it indicates impaired absorption, which may be due to either lack of IF or small intestinal malabsorption Part 2 performed if part 1 of test is abnormal

42. Part 2 : patient is orally administered radiolabelled vitamin B 12 along with IF while remainder of test is carried out out as in part 1 Excretion becomes normal – lack of IF Excretion remains below normal – defective absorption in small intestine

43. 4. INTRINSIC FACTOR ANTIBODIES IN SERUM Detection of anti-IF antibodies in serum is diagnostic of pernicious anemia

44. MANAGEMENT OF B 12 DEFICIENCY When B 12 deficiency is suspected a trial of B 12 is essential. Failure of response can only be determined after careful follow-up over a period of several months, particularly if the patient is non-anaemic. Standard therapy for all cases of B 12 deficiency is by regular intramuscular injections of B 12 , usually in the form of hydroxycobalamin. In patients with inadequate dietary intake supplements may be given by mouth. Underlying conditions should be managed separately.

45. After initiation of therapy, reticulocyte count begins to increase around 3 rd day – peak by 6 th or 7 th day – gradually returns to normal by end of 3 rd week Hematocrit steadily rises and normalises in about 1-2 months Blood transfusion is indicated in severely anaemic symptomatic patients or in patients with CCF

46. NOTE: Both B 12 and folate are given to patients if B 12 deficiency has not been excluded. This is to prevent neurological damage, e.g. subacute combined degeneration of the spinal cord.

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