Megaloblastic Anaemias PDF

Megaloblastic Anaemias Dr Nina Dempsey-Hibbert  \ MEGA NORM MICRO \ \ Macro \  Megaloblastic Anaemias: What are they? * Group of anaemias in which there is delayed maturation of the ERYTHROBLAST nucleus in relati...

Megaloblastic Anaemias Dr Nina Dempsey-Hibbert  \ MEGA NORM MICRO \ \ Macro \  Megaloblastic Anaemias: What are they? * Group of anaemias in which there is delayed maturation of the ERYTHROBLAST nucleus in relation to the cytoplasm * *Erythroblast – immature – still retains a nucleus * Asynchronous maturation of nucleus due to defective DNA synthesis – seen in the bone marrow * Presents as MACROCYTIC anaemia  What causes Megaloblastic Anaemia? * Usually deficiencies of either vitamin B12 or Folate * May also occur due to * Abnormalities of vitamin B12 or Folate metabolism * Defects of DNA synthesis - rare * Vitamin B12 and Folate are crucial in DNA synthesis  Vitamin B12 * Vitamin B12 (Cobalamin) is synthesised by micro-organisms – animals must consume it through diet * Only contained in food of animal origin - 50% young adult vegans have subnormal B12 levels * Liver is the major storage site * Body stores 3-4yrs worth – short term dietary deficiency not problematic  B12 Absorption Salivary glands secrete B12 is consumed R-PROTEIN with dietary proteins Parietal cells in stomach secrete INTRINSIC FACTOR (IF) Chief cells in stomach secrete PEPSIN B12 Absorption PEPSIN catabolises dietary proteins and separates the B12 B12 Absorption R-PROTEIN (from the mouth) binds free B12  B12 Absorption B12, R-PROTEIN and IF move into Duodenum Pancreas starts to secrete digestive enzymes (lipases, proteases etc.)  B12 Absorption Pancreatic proteases catabolise R-PROTEIN and free up the B12  B12 Absorption Free B12 is now able to bind to IF  B12 Absorption * B12 must be attached to IF for it to be absorbed * Receptors on enterocytes only recognise the B12-IF complex * Additionally, IF protects the B12 from catabolism by intestinal bacteria ** approx. 1% of B12 can be absorbed in the absence of IF**  ENTEROCYTES B12 IF B12 IF B12 IF TERMINAL ILEUM B12 IF B12 IF IF B12 B12 IF IF IF – Intrinsic Factor B12 Absorption B12 – IF complexes are absorbed at the terminal ileum  Vitamin B12 * Once absorbed, B12 passes into the portal blood * Attaches to TRANSCOBALAMIN which delivers B12 to the bone marrow and other tissues * **Megaloblastic anaemia can also result from transcobalamin deficiency – but Vitamin B12 intake is normal**  TC TC B12 PORTAL BLOOD TC B12 TC B12 ENTEROCYTES B12 IF B12 IF B12 IF IF B12 TERMINAL B12 IF ILUM IF IF B12 IF B12 IF – Intrinsic Factor TC- Transcobalamin  Vitamin B12 * Once in the bone marrow/tissues, the B12-transcobalamin complex binds to a receptor and is endocytosed * B12 is then able to perform its function as co-factor in crucial biochemical pathways nucleus nucleus TC B12 R R TC B12  Folate * Humans are unable to synthesise folate * Require pre-formed folate in the diet – mostly in green leafy vegetables *foliage!* * Liver is major storage site  Folate requirements * Dietary folates are absorbed as methyl tetrahydrofolate (methyl THF) in the upper small intestine * Folate binding proteins are present on cell surfaces to facilitate entry into the cell * Once inside the cell they are converted to folate polyglutamates * Faster turnover - folate deficiency develops much faster than B12  DNA dATP dGTP dCTP dTTP dTDP DHF THF polyglutamate dTMP polyglutamate 5,10-methylene THF polyglutamate dUMP THF Methionine Methyl THF Homocysteine CELL Methyl THF PLASMA  Folate and B12 * Vitamin B12 is an essential co-factor for the enzyme Methionine synthetase, needed for the conversion of methyl THF to THF * This reaction also involves the methylation of homocysteine to form methionine  DNA Act as folate co-enzymes in the conversion of dUMP to dTMP dATP dGTP dCTP dTTP dTDP DHF THF polyglutamate dTMP Rate polyglutamate limiting 5,10-methylene THF step polyglutamate dUMP THF Methionine Methyl THF Homocysteine CELL Methyl THF PLASMA  Lack of B12 prevents demethylation DNA of methyl-THF, preventing formation of THF polyglutamates dATP dGTP dCTP dTTP dTDP DHF THF polyglutamate dTMP Rate polyglutamate limiting 5,10-methylene THF step polyglutamate dUMP THF Methionine Vitamin B12 deficiency X Methionine Synthase Methyl THF Homocysteine CELL Methyl THF PLASMA Lack of folate prevents formation DNA of THF polyglutamates dATP dGTP dCTP dTTP dTDP DHF THF polyglutamate dTMP Rate polyglutamate limiting 5,10-methylene THF step polyglutamate dUMP THF Methionine Methionine Synthase Methyl THF Homocysteine CELL Folate X Methyl THF deficiency PLASMA  Folate/B12 Deficiency * Deficiencies in either B12 or folate therefore result in a decrease in DNA synthesis DELAYED MATURATION OF THE NUCLEUS IN RELATION TO THE CYTOPLASM * As there is a high turnover of cells in the bone marrow, this site is where the problems become obvious  Megaloblastic Anaemia * So, we know that both vitamin B12 and Folate have crucial roles in DNA synthesis……. * therefore deficiency of EITHER B12 or Folate causes MEGALOBLASTIC anaemia  Clinical Features y pical of T p toms sym aemia an * Patients complain of: * Tired all the time (TATT) * Shortness of breath (particularly on exercise) * Weakness  Clinical Features * Pallor of mucous membranes * Slight jaundice * Glossitis * Nervous system disturbances - impaired vision, gait disturbance * Psychiatric disturbances – mood swings, irrational behaviour * Many patients may be asymptomatic - diagnosed following routine blood test - reveals macrocytosis  **Laboratory Findings** Blood Macrocytosis – therefore MCV > 95fL ** macrocytosis is the earliest sign of B12 deficiency and can be detected even before the onset of anaemia Macrocytes are typically oval in shape - ovalocytes  **Laboratory Findings** RBCs larger than normal… but some Normal RBCs RBCs normal size: are approx. same size as ANISOCYTOSIS the nucleus of a lymphocyte Oval-shaped RBCs  **Laboratory Findings** * Hypersegmented neutrophils are one of the hallmarks of megaloblastic anaemia * 6 or more lobes - R Shift * This results from delayed DNA synthesis but intact segmentation mechanism Normal Neutrophil Right-shifted Neutrophil  **Laboratory Findings** POIKILOCYTOSIS HOWELL-JOLLY BASOPHILLIC BODY STIPPLING Abnormal shaped Aggregation of Nuclear remnants ribosomal RNA RBCs  **Laboratory Findings** * Increase in serum un-conjugated bilirubin - due to marrow cell breakdown * Increase in lactic acid dehydrogenase (LDH) – due to marrow cell breakdown * Normal serum iron and ferritin  **Laboratory Findings** Not always so easy to diagnose 25-40% with B12 deficiency have normal MCVs same % have normal Hb on presentation  Serum Vitamin B12 * Measured by commercial fully automated assays * Will be low in cases of B12 deficiency (some exceptions) * Will be normal (or borderline) in cases of folate deficiency * Not considered a robust assay  Serum Folate * Will be low in cases of folate deficiency * Will be normal or raised in cases of B12 deficiency  Red Cell Folate * Will be low in folate deficiency * Not specific to folate deficiency - 60% B12 deficiency cases have low red cell folate * More reliable guide of tissue folate status than the serum folate test  Homocysteine & Methylmalonic Acid Serum Homocysteine increased in both B12 and For ic l Folate deficiency Top ctica Pra sion! Ses Serum methylmalonic acid (MMA) increased in B12 deficiency only Normal levels of both MMA and homocysteine rule out clinically significant B12 deficiency with virtual certainty Overview of Investigations Clinical History – signs of anaemia Blood test Macrocytic cells LDH and observed on blood MCV Increased unconjugated smear bilirubin increased Further Investigations Serum B12 Low Serum Folate Serum methylmalonic Low acid & homocysteine increased B12 Deficiency Folate Deficiency B12 Deficiency  Next Step – why?? * Once a deficiency has been confirmed, it is important to determine the cause…. Dietary Cause? Lack of IF/parietal cell dysfunction Pathological cause Protease or in terminal ileum pancreatic insufficiency  Causes of B12 Deficiency Lack of B12 absorption Transcobalamin Deficiency Congenital lack of Intrinsic Adequate B12 absorption factor* but poor transport Congenital lack of B12-IF receptors* Autoimmune attack on gastric mucosa, IF or parietal cells (Pernicious Anaemia) *will cause B12 deficiency in childhood but not until all B12 stores derived from the mother ‘in utero’ are all used up (i.e. 2yrs)  Causes of Folate Deficiency Poor dietary intake Chronic diseases of digestive tract – interfere Pregnancy – increased with absorption demand Coeliac Crohn’s Long term use of antibiotics Tropical sprue-disease Interfere with normal absorption Gastric intestinal surgery Drugs - antagonists of folate metabolism E.g. Methotrexate  B12 Absorption Test * For years, the gold standard test to distinguish between malabsorption and poor dietary intake was the SCHILLINGS TEST * Uses radiolabelled B12 to measure absorption of free or IF- bound B12 * The test is now becoming rare due to increasing difficulties in obtaining the isotope  Schilling Test – Step I Patient given Intramuscular B12 – this will move directly to the liver – saturate all B12 receptors in the liver Patient also given oral dose of radiolabelled B12 (57Co-labelled) Radiolabelled B12 SHOULD be absorbed in the intestine, move to the liver – but liver is saturated Radiolabelled B12 SHOULD be excreted in the urine over 24h period If radiolabelled B12 is present in the urine – dietary problem  Schilling Test – Step II If results from step I show no radiolabelled B12 in the urine – suggests absorption problem Next - Radiolabelled B12 plus intrinsic factor is administered Urine is monitored over 24 period If radiolabelled B12 is present in urine – indicates IF deficiency – most commonly caused by pernicious anaemia**  Schilling Test – Step III If results from step II show no radiolabelled B12 in the urine – suggests less common cause Next - Radiolabelled B12 plus antibiotic is administered Urine is monitored over 24 period If radiolabelled B12 is present in urine – indicates bacterial overgrowth in terminal ileum impeding B12 absorption  Schilling Test – Step IV If results from step III show no radiolabelled B12 in the urine… Radiolabelled B12 plus pancreatic enzymes are administered Urine is monitored over 24 period If radiolabelled B12 is present – indicates pancreatic enzyme insufficiency – R-protein remains bound to B12 – therefore IF is unable to bind – no absorption by enterocytes e.g. chronic pancreatitis  Schilling Test * Remember…… whatever causes the presence of radiolabelled B12 in the urine, is what was originally deficient: Add B12 only Poor B12 intake Add IF IF deficiency Add antibiotics Bacterial overgrowth Add Pancreatic Pancreatic Enzymes insufficiency  B12 and Folate independent mechanisms * It is important to know that Megaloblastic anaemia can also result from mechanisms other than B12 and Folate deficiency * Drugs affecting nucleic acid synthesis - Chemotherapy drugs e.g. Azathioprine, hydroxycarbamide * Inherited causes  Treatment…. Depends on the Cause B12 Poor Intake Change in diet and/or oral supplementation – concomitant folic acid often given Other Causes – Lifelong treatment High-dose oral supplementation **remember small proportion of B12 absorption is independent of B12 OR Intramuscular injections of B12 – initial high loading dose (5x 1000mg) then 3- monthly injections Deficiency in digestive enzymes may be treated separately Folate All causes Change in diet and/or oral supplementation  Important Points * What is MEGALOBLASTIC Anaemia? * What causes it? * What are the morphological features that can help diagnosis? * What is the Shilling Test?  Reading * McGraw-Hill - Haematology in Clinical Practice * Chapter 8 – “Macrocytic Anaemias” * Common morphological findings in anaemias – Chapter 2 “Clinical Approach to Anaemia”