MEGALOBLASTIC ANAEMIAS

MEGALOBLASTIC ANAEMIAS

Introduction ..Characterized by defective synthesis of deoxyribonucleic acid (DNA) in all proliferating cells

Most commonly result from lack of folic acid or vitamin B12

MEGALOBLASTIC ANAEMIA[ VITAMIN B12 DEFICIENCY ]

Normal Vitamin B12 MetabolismVitamin B12 is composed ofA corrin nucleus which has 4 pyrrole rings bound to a central cobalt atomA 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

Sources Liver, dairy products and seafish are major sourcesAlthough bacteria in the large intestine synthesize vitamin B12 it cannot be absorbed from this siteMinimum amount required for an adult is 1 to 4 g per day

Absorption of Vitamin B122 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 B12 are ingested

Vitamin B12 in foodR-BinderB12-R-Binder complexIF-B12 complex + Freed R-BinderIntrinsic Factor (IF)Receptor-IF-B12B12-TCIICirculation

ReceptorTCIIIFDegradationEpithelial cell of terminal iIeum

StomachDuodenum

Transport of Vitamin B12Following absorption by the ileal mucosal cells, vitamin B12 is carried in the plasma by various transporting proteins:

Transcobalamin I Transcobalamin IITranscobalamin III

Transcobalamin I (TC I) is an alpha-globulin produced by granulocytes. It functions as a circulating reserve store of B12. TC I carries mostly methylcobalamin.

Transcobalamin II (TC II) is a beta-globulin formed in the liver and is the dominant carrier of B12 immediately after absorption. It is the main agent for rapid transport of B12 to the body cells.

Transcobalamin III (TC III) is an alpha-globulin. TC III may act as a defence mechanism by depriving pathogens of B12 at sites of infection

Storage sites Total amount of vitamin in body is 2-5 mg ( adequate for 3 years ) Major site : liverExcreted through the bile and shedding of intestinal epithelial cells Most of the excreted vitamin B12 is again absorbed in the intestine (enterohepatic circulation)

Functions of Vitamin B12

Synthesis of methionine from homocysteineConversion of methyl malonyl CoA to succinyl CoA

FH4FH2

Methylene FH4Methyl FH4

Intestinal cellDietary folatesDihidrofolateRedutaseMethionineHomocysteineThymidylate SynthaseDNA SynthesisdTMPdUMPRole of Vitamin B12 and Folate in DNA synthesis

VitB12 (Methylcobalamin)

General Morphological Features Of Megaloblastic Anemia

PERIPHERAL BLOOD FINDINGSHemoglobin decreasedHematocrit decreasedRBC count decreased/normalMCV - >100fl ( normal 82-98fl)MCH increasedMCHC NORMALReticulocytopenia.Total WBC count normal / lowPlatelet count normal/ lowPancytopenia, especially if anaemia is severe.

PERIPHERAL SMEARRBC:- Macro ovalocytes (macrocytic normochromic)[ macrocytosis is the earliest sign in Vit B12 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, Cabots rings may be found

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

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

PERIPHERAL SMEARWBCNormal count or reduced countHypersegmented 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

BONE MARROWMarkedly hypercellularMyeloid : 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

MEGALOBLASTCell and nuclear size and amount of cytoplasm (deeply basophilic royal blue) are increased Nuclear chromatin is sieve like or stippled (open)Nuclear cytoplasmic asynchrony/dissociationAbnormally large precursor (promegaloblast and early megaloblast) are increased in BM Maturation arrestAbnormal mitoses (increased)

Granulocytic series also display megaloblastic changesMost 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

BIOCHEMICAL FINDINGS

Increase in serum unconjugated bilirubin- because of ineffective erythropoiesisIncrease is LDHNormal serum iron and ferritin

Causes of Vit B12 deficiency

Insufficient dietary intake (very rare)Strict vegetarians Deficient absorptionPernicious anaemiaTotal or partial gastrectomyProlonged use of PPI or H2 blockersDiseases of small intestine Fish tapeworm infestation

PERNICIOUS ANEMIA

Thomas Addison (1849)Disease of elderly 5th to 8th decades (median age at diagnosis 60 years)Genetic predispositionTendency to form antibodies against multiple self antigens

PATHOGENESISImmunologically mediated, autoimmune destruction of gastric mucosaCHRONIC ATROPHIC GASTRITIS marked loss of parietal cells

Three types of antibodies:Type I antibody- 75% - blocks vitamin B12 and IF bindingType II antibody prevents binding of IF-B12 complex with ileal receptorsType III antibody 85-90% patients against specific structures in the parietal cell

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

Associated with other autoimmune disorders like Hashimotos, Graves, vitiligo, diabetics mellitus, primary hyperparathyroidism, Addisons and Myasthenia gravis

Patients with pernicious anaemia have increase risk of gastric cancer

DIAGNOSTIC FEATURESModerate 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 B12

Elevated levels of homocysteine

Striking reticulocytosis after parenteral administration of vitamin B12

Serum antibodies to intrinsic factor (specific) and anti parietal cell antibodies in serum

Abnormal Schilling test, pentagastrin-fast achlorhydria

GASTRECTOMY

Total gastrectomy :Secondary to Vit B12 deficiency as it removes the site of synthesis of intrinsic factorProphylactic vitamin B12 after surgeryPartial gastrectomyRegular follow up after surgery for early detection of deficiency

DISEASES OF SMALL INTESTINETuberculosis, whipples disease, blind loop syndrome or resection of small intestine may interfere with absorption that occurs in the terminal ileumBlind loop syndrome stasis of small intestine contents (diverticulum / stricture) may predispose to bacterial colonization and proliferationUtilization of most of the ingested Vit B12 by bacteria may lead to reduced or non avail of Vit for absorption

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

CLINICAL FEATURES

Anaemia, mild jaundice and sometimes neurological involvementNeurological involvement in the form ofPeripheral neuropathySubacute combined degeneration of spinal cordCerebral changes (personality changes, dementia & psychosis)

Patients can present with only neurological abnormalities without megaloblastic anaemia

LABORATORY FEATURES

Morpholgical changes of megaloblastic anaemia in PS and BMSerum vitamin B12 assaysMethylmalonic acid (MMA) and homocysteine in serumSchilling testIntrinsic factor antibodies in serum

1. SERUM VITAMIN B12 ASSAYS

Various methods are available, e.g. microbiological methods using Lactobacillus leichmannii or radio-isotope techniques (RIA) using 57CoB12, coated charcoal and IF.

RADIO-ISOTOPE DILUTION ASSAY:

A known amount of radioactive (hot) B12 is diluted with the non-radioactive (cold) B12 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 B12. The bound B12 is separated from the free and its radioactivity counted.

The count is inversely proportional to the B12 concentration in the test serum.

The higher the serum B12 the greater will be the dilution of the radioactive B12 and thus less radioactivity attached to the IF.

By comparison with standards of known B12 content, the B12 content of the test serum can be calculated.

In Vitamin B12 deficiency ,Serum Vitamin B12 and red cell folate are depressedSerum folate is normal or increased ( accumulation of 5-methyl tetrahydrofolate ) [folate trap]

2. Methylmalonic acid (MMA) and homocysteine in serumRecent reports of S.methylmalonic acid and S.homocysteine are more sensitive for detection of Vitamin B12 than estimation of Vitamin B12

Raised early in tissue deficiency even before appearance of hematological changes

3. SCHILLING TESTFor evaluation of absorption of vitamin B12 in the GIT

Performed in 2 parts part 1 and part 2

Part 1 : 0.5 to 1 g of radiolabelled vitamin B12 is given orally After 2 hrs IM dose (1000 g) of unlabelled vitamin B12 is given [ saturates binding sites of TC I and TC II and displaces any bound radiolabelled vitamin B12 (thus permitting urinary excretion of absorbed radiolabelled vitamin B12 )

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 B12 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

Part 2 : patient is orally administered radiolabelled vitamin B12 along with IF while remainder of test is carried out out as in part 1

Excretion becomes normal lack of IFExcretion remains below normal defective absorption in small intestine

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

MANAGEMENT OF B12 DEFICIENCY

When B12 deficiency is suspected a trial of B12 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 B12 deficiency is by regular intramuscular injections of B12, usually in the form of hydroxycobalamin. In patients with inadequate dietary intake supplements may be given by mouth. Underlying conditions should be managed separately.

After initiation of therapy, reticulocyte count begins to increase around 3rd day peak by 6th or 7th day gradually returns to normal by end of 3rd 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

NOTE:

Both B12 and folate are given to patients if B12 deficiency has not been excluded.This is to prevent neurological damage, e.g. subacute combined degeneration of the spinal cord.

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