MACROCYTIC ANAEMIA

INTRODUCTION
 Macrocytic anaemias are characterized by large erythrocytes (average mean corpuscular volume more than 100fl ) that usually have normal haemoglobin content in relation to their size.  Macrocytosis is frequently a sign of disease process that can result in significant morbidity if left untreated .

 In 60% cases macrocytosis is not accompanied by anaemia ; however isolated macrocytosis should be always investigated.  Macrocytosis without anaemia may be an indication of early folate or cobalamine deficiency ,as macrocytosis precedes development of anaemia.

 Causes of artifactual macrocytosis when blood is analyzed on on automated instruments  cold agglutinins  hyperglycemia  marked leukocytosis  MOST COMMON CAUSE OF MACROCYTOSIS is  alcoholism followed by folate & cobalamin deficiency .

CLASSIFICATION
BASED ON MORPHOLOGICAL CHARACTERISTIC OF MEGALOBLASTIC Abnormal DNA synthesis( nuclear maturation defect) Oval macrocytes Causes-Vitamin B12 & folate deficiency(95% cases) Chemotherapy ,myelodysplasia & other primary marrow tumors ERYTHROID PRECURSORS IN BONEMARROW NONMEGALOBLASTIC not well defined : may be due to increase in memberane lipids Round macrocytes Causes-Alcoholism Liver disease, haemolysis or bleeding Hypothyroidism.

MEGALOBLASTIC ANAEMIA
Characterised by distinctive cytological and functional abnormalities in peripheral blood and bone marrow cells due to nuclear maturation defect with anaemia primarily attributed to a large degree of ineffective erythropoiesis.

 The anaemia was called megaloblastic so as to describe the giant abnormal appearing erythroid precursors( megaloblasts) in the bone marrow. Paul Ehrlich is credited with coining the term megaloblasts used to describe the large abnormal precursors.

VITAMIN B 12

VITAMIN B12 MOLECULE

Structurally classified as a corrinoid. Molecule is composed of three portions 1. A corrin ring with four pyrrole group and a cobalt at the centre. 2. A nucleotide which lies perpendicular to the ring & attached to the ring and cobalt. 3. A beta group attached to cobalamine on the opposite side of the ring from the nucleotide.

METABOLISM.. ABSORPTION

TRANSPORT

FUNCTION

Pathophysiology of cobalamine deficiency

Impaired DNA synthesis lack of methionine synthetase >>>> defect in thymidylate synthesis>>>> defect in DNA synthesis. All dividing cells are affected>>> megaloblastic anaemia & columnar and squamous epithelial cells abnormalities.

 Defective fatty acid degradation- Demyelination of nerves due to defect in degradation PROPIONYL coA Primer for FA synthesis
Adenosylcobalamine

FA with odd number of C>> incorporated into neuronal membranes>> disruption of membranes Neurologic disease

SUCCINYL co A

REQUIREMENTS
Diet: 5 7 ug / day Needed: 3 5 ug / day

STORAGE
Storage form; adenosylcobalamine Max in liver and then in heart and kidney

Vitamin B12 deficiency symptoms include: Pallor, weakness, fatigue and tachycardia. Palpitations. Loss of appetite. Difficulty in concentration and irritability. In severe cases, symptoms of what is called "Sub-acute Combined Degeneration" can occur and they include:

Numbness and tingling in the peripheries. Tingling and pain in the palm of the hands and the tongue. Impaired sensations and maybe impaired equilibrium. Abnormal reflexes. Atrophy in the peripheries

CAUSES OF VITAMIN B12 DEFICIENCY

Inadequate intake strict vegetarians Malabsorption- pernicious anaemia, gastrectomy, coeliac dis. ,tropical sprue, crohns dis. , tuberculosis,ileal resection, drugs , fish tapeworm infestation Impaired utilization-transcobalamin ll deficiency nitrous oxide inhalation, inborn error of metabolism Increased demand- pregnancy, neoplasms,chronic haemolytic anaemia Myeloproliferative disorders, hyperthyroidism

PERNICIOUS ANAEMIA
Autoimmune disease Decreased absorption of vitamin B-12 from the gastrointestinal tract in pernicious anemia results from the presence of an autoantibody against intrinsic factor (IF), a protein made in the stomach that is necessary for the absorption of vitamin B-12 It is associated with other autoimmune diseases like addison disease , thyroiditis, vitiligo

FOLIC ACID

STRUCTURE OF FOLATE
Chemical name- PTEROYL GLUTAMIC ACID Structurally- 3 parts: 1. Pteridin, a nitrogen containing ring. 2. A ring of paraaminobenzoic acid. 3. A chain of glutamic acid residues. It is the inert form of folate. THF active form of folate is formed by a four hydrogen reduction of pteridine ring. Function of THF is to transfer carbon units from donors to acceptors.

 Most Folic Acid in food is in the conjugated polyglutamate form. It is deconjugated in intestine to the monoglutamate form. Absorption takes place through out small intestine but esply in proximal jejunum.

 Once absorbed into the intestinal epithelial cells the folate is reduced to N5 Methyl THF (Primary circulating form of THF in blood stream)>>>> attaches to cells by means of specific receptor. Once inside the cell it must be demethylated and conjugated to keep it from leaking out again. Demethylation reaction requires cobalamine. Deficiency of Vitamin B 12 >>>> TRAP FOLATE IN METHYLATED FORM blocking the formation of conjugated THF.>>>> The cells are unable to retain their folate leading to tissue folate depletion.

FUNCTION
vital role in metabolism of nucleotides & aminoacids

Pathophysiology of folate deficiency

REQUIREMENT
Recommended daily dietary allowance for adults -200 micrograms Minimum daily requirement50microgram/day Storage in liver -5 10 mg of folate .

FOLATE DEFICIENCY
Irritability or Depression Tongue changes - glossitis High Homocysteine Level-People with high homocysteine levels have been shown to have a higher increase in cardiovascular events such as heart attacks. Birth Defects and MiscarriageFolic acid is critical in the development of your fetus. Cancers Diarrhea and loss of appetite

CAUSES OF FOLATE DEFICIENCY

Inadequate dietary intake (90% cases)nutritional deficiency Malabsorption- coeliac disease ,tropical sprue Drug induced- methotrexate, OCP, anticonvulsants, cholestyramine Inherited enzyme deficiency-DHFR , NMTHFT,FIT Increased requirement- pregnancy ,lactation ,haemolytic anaemia, prematurity ,myeloproliferative disorder, neoplasms, chronic inflammation, hyperthyroidism

MISCELLANEOUS CAUSES OF MEGALOBLASTIC ANAEMIA

Congenital disorders of DNA synthesisorotic aciduria methionine synthase deficiency congenital dyserthropoietic anaemia homocystinuria,methylmalonic aciduria Acquired disorders of DNA synthesismyelodysplastic syndrome acute erythroblastic leukaemia metabolic inhibitor drugs- methotrexate, hydroxyurea ,cytosine arabinoside refractory sideroblastic anaemia

Diagnosis of megaloblastic anaemia

LABORATORY FINDINGS: Haematological parameters vary considerably.  Megaloblastic anaemia involves all 3 blood cell lines : erythrocytes, leukocytes, platelets.  Haemoglobin- normal to very low  Erythrocyte count - normal to very low  Leukocyte count - decreased (absolute neutropenia)  Platelet count - decreased  Relative reticulocyte count normal  RPI less than 2  Indices- MCV- increased , MCH-increased, MCHCnormal

PERIPHERAL BLOOD FILM

Anisocytosis- moderate to severe with normocytes & few TRAID OF FEATURES microcytes in addition to OVAL MACROCYTES macrocytes. Poikilocytosis- severe with severe anaemia HOWELL-JOLLY BODIES Polychromatophillia & nucleated RBCs HYPERSEGMENTED RBCs may show basophillic stippling , cabot ring NEUTROPHILS WBCs- neutrophil show hypersegmentation & mild shift to left may be noted Platelets may be enlarged

Oval Macrocyte Howell Jolly Body Hypersegmented neutrophils

Basophillic stippling
Cabot ring Nucleated RBCs

Hypersegmented neutrophil

Polychromasia

BONE MARROW MORPHOLOGY

 CELLULARITY:- hypercellular with an increase in erythroid precursors  M:E ratio:- decreased  ERYTHROPOIESIS:- megaloblastic changes occur at all stages of red cell development. MEGALOBLASTS ARE LARGE NUCLEATED ERYTHROID PRECURSORS WITH NUCLEAR MATURATION LAGGING BEHIND CYTOPLASMIC MATURATION  MATURATION:- maturation arrest promegaloblasts & basophillic megaloblasts constitue more than 50% of erythroblasts

 NUCLEUS:-chromatin network is more open , being arranged in a fine reticular fashion to give a stippled appearance - well marked in polychromatic cells , & is sometimes seen in orthochromatic cells . -nucleus of orthochromatic cell is indented or lobulated with one/more howell- jolly bodies.  DISSOCIATION OF CYTOPLASMIC & NUCLEAR MATURATION :- the nucleus is younger than normal appearance for the apparent , more mature stage of the cytoplasm . Cytoplasm development (haemoglobinization) occurs at normal rate ( NUCLEUS- CYTOPLASMIC ASYNCHRONY)  MITOSIS:- common

Erythroid hyperplasia

megaloblasts

 Prussion blue staining of marrow shows

increase in number & size of iron granules in erythroid precursors .
..

 LEUCOPOIESIS:-absolute number of developing granulocytes is increased but their % is decreased. - presence of large atypical granulocytes , occur at all stages of development especially metamyelocyte stage GIANT STAB / giant metamyelocye 30micron in diameter & large U-shaped nucleus . BAND forms with loose open chromatin.

 MEGAKARYOPOIESIS:-present in normal/ increased / decreased number. -Some are atypical & have deep basophillic granular cytoplasm or hypersegmented nucleus.

giant metamyelocyte

OTHER LABORATORY FINDINGS

 Increase in plasma iron turnover serum iron indirect bilirubin urobilinogen LDH1 & 2 FIGLU excretion (after administration of histidine)  Decrease in haptoglobin uric acid alkaline phosphatase  Specific tests for vitamin B12 & folate include assays of serum levels.

 Special tests to diagnose vitamin B12& folate deficiency SERUM vitamin B12 assay microbiological radio-isotope MICROBIOLOGICAL ASSAY Principle the serum to be assayed is added as a source of vitamin B12 to a medium containing all other essential growth factors for vitamin B12 dependent microorganism (lactobacillus leishmanii & Euglena gracilis ) The medium is then inoculated with the organism , the amount in serum is determined by comparing the growth as estimated turbimetrically with the growth produced by standard amount of vitamin B12.

 RADIO-ISOTOPE ASSAY isotope dilution of nonradioactive serum vitaminB12 by adding co57- labelled B12. & a vit. B12 binding protein .

Normal serum B-12 levels 200-1000pg/ml

 False increase CML, liver disease , polycythemia rubra vera, transcobalamine 2 deficiency, nitrous oxide anaesthesia  False decrease severe folate deficiency ,pregnancy, multiple myeloma, veg. diet , R-protein deficiency , trancobalamin 1 deficiency

 Serum & erythrocyte folate assay

Microbiological radio-isotope MICROBIOLOGICAL lactobacillus casei ( reference range is 3-25ng/ml

RADIO-ISOTOPE uses pteroylglutamic acid or methyltetrahydofolate & a folate binding protein (R.r-210ng/ml)
 Erythrocyte folate level gives better indication of folate stores.

 The diagnosis of folate and vitamin B12 deficiencies may be confusing. With cobalamindeficiency, serum cobalamin levels are usually low, but many are normal. The serum folate level is very sensitive to folate intake, and a recent folate-rich meal can normalize it. Red cell folate measurements, which can better reflect tissue levels, have several problems ( false increase & decrease).

 measure homocysteine, which increases in both disorders because methionine synthesis is impaired by deficiency of either. This laboratory finding typically precedes decreases in serum levels of folate and cobalamin.

A cobalamin-dependent, but folate-independent, enzymatic reaction leads to increased serum levels of methylmalonic acid (MMA) with cobalamin deficiency. Accordingly, measurement of both homocysteine and MMA reliably detect, and distinguish between, folate and cobalamin deficiencies.

 When both are elevated, cobalamin deficiency is confirmed, although concurrent folate deficiency is possible. If homocysteine is elevated and MMA is normal, folate deficiency is likely. If both are normal, deficiency of either is highly improbable.

Specific tests for pernicious anaemia Gastric analysis if there is no free

HCl after histamine stimulation, this may indicate PA since the same cells that secrete HCl, also secrete intrinsic factor (IF)

Test for antibodies

antibodies to parietal cells(not specific) Antibodies against IF(specific)

Schilling test is the definitive test for the diagnosis of PERNICIOUS ANAEMIA

 The test measures the amount of an oral dose of radioactively labeled B12 that is absorbed in the gut and excreted in the urine. This is followed by an injection of unlabeled vitamin B12 to saturate all vitamin B12 receptors in the tissue and plasma. Thus any amount absorbed in the gut will be in excess, and will be filtered in the kidneys to appear in the urine. If there is no radioactivity in the urine, this means that there is either malabsorption or pernicious anaemia The test is repeated, but this time the radioactively labeled B12 is accompanied by a dose of IF. If absorption is now normal, this means that the patient has PA if not this means malabsorption .

NON MEGALOBLASTIC MACROCYTIC ANAEMIA

Macrocytic RBCs are not oval, but are round.  No hypersegmented neutrophils or Howell-Jolly bodies

LAB RESULTS OF MEGALOBLASTIC VS NON-MEGALOBLASTIC MACROCYTOSIS

DIFFERENTIAL DIAGNOSIS WITH INCREASED MCV

TREATMENT
To treat the underlying cause
eg: To determine which deficiency exists &treat the patient with the specific vitamin.  pernicious anaemia- lifelong monthly parentral doses of hydroxycobalamin

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