MACROCYTIC ANEMIAS Ahmad Sh MACROCYTIC ANEMIAS Ahmad Sh. Silmi Staff Specialist in Haematology Medical Laboratory Sciences Dept, IUG 2012

Introduction Anemia classification based on the mechanism Kinetic Classification (based on retic count) Decreased production Morpholgical classification (based on MCV) Microcytic Normocytic Macrocytic Increased destruction Immunological classification (based on Coomb’s test) Immune-mediated Non-immune mediated

The Medical Student’s Approach to Anemia Check the reticulocyte count to determine if the anemia is from decreased production (“hypoproliferative”, “reticulocytopenic”) or increased destruction (“hemolytic”)/acute blood loss (“reticulocytosis”) 2. If decreased production, narrow down the causes in terms of the MCV- If the MCV is low, then do iron studies then Hb electropheresis If the MCV is normal, check the serum creatinine and TSH, if they are WNL then consider bone marrow exam If the MCV is high check a folate and vitamin B12 level 3. If the reticulocyte count is increased- Check a direct Coomb’s test 4. Look at the peripheral blood smear to confirm/support the diagnosis

Big Issue‼ MCV=mean corpuscular volume

Renal vs. Liver vs. Endocrine vs. Anemia of Inflammation Anemia Algorithm Patient with anemia and decreased reticulocyte count- What is the MCV ?? Microcytic Normocytic Macrocytic: Vitamin-related B12, Folate Non-vitamin: MDS EtOH/Liver Disease Hypothyroidism Diseases in Bone Marrow MDS Solid Tumor Myeloma Aplastic anemia Fe def. Systemic Diseases Thal Renal vs. Liver vs. Endocrine vs. Anemia of Inflammation Other: sideroblastic anemia

Anemia : MCV >100 80-100 <80 Macrocytic Normocytic Microcytic Defective DNA synthesis Megaloblastic anemia B12 deficiency Folate deficiency Myelodysplastic Syndromes Young RBCs-reticulocytosis Hemolytic anemia Hypoplastic anemias Aplastic anemia 80-100 Normocytic Decreased production Aplastic anemia Increased destruction Acute blood loss Hemolytic anemias Acquired Autoimmune HA Infectious disease Inherited Membrane defects Hereditary spherocytosis Hereditary elliptocytosis Enzyme defect/deficiency Glucose 6 phosphate dehydrogenase Pyruvate kinase Unstable hemoglobin <80 Microcytic Defective hemoglobin synthesis Iron deficiency (Fe2+) Thalassemia (globin chain) Sideroblastic anemia (heme) Low Reticulocyte index High

Requirements for Red Blood Cell Production Erythropoeitin Proteins, required for globin synthesis Iron Vitamin B12 and folic acid Vitamin B6 Vitamin C Thyroid hormones, estrogens and androgens

Definitions Macrocytic MCV > 100 MCH is increased due to increased cell size MCHC is normal as concentration of hemoglobin is normal Macrocytosis is found in 2.5-4% of adults having a routine CBC Sub classification Megaloblastic Non-megaloblastic

Macrocytic anemias Megaloblastic Non-Megaloblastic More severe macrocytosis with oval erythrocytes Usually MCV is >110 Defects in DNA syntheses leading to delayed nuclear development in the face of normal cytoplasmic development Marrow shows nuclear cytoplasmic asynchrony Erythroid lineage Myeloid lineage Reticulocyte index is not increased Non-Megaloblastic Less severe macrocytosis with round erythrocytes Usually MCV is <110 Pathophysiology unknown Increase in membrane lipids Immature cells (reticulocytes) are increased

Macrocytic anemias * * * Megaloblastic Non-Megaloblastic B 12 deficiency Folate deficiency Drugs Megaloblastoid morphologic changes can be seen in Myelodysplastic syndromes Congenital dyserythropoietic anemias Non-Megaloblastic Alcoholism Liver disease Hypothyroidism Aplastic anemia Hemolysis, acute bleed Increased reticulocytes and immature erythrocytes Artifact RBC clumping cold agglutinin disease Hyperglycemia RBC swelling * * *

MEGALOBLASTIC ANEMIA These are a group of disorders in which the cause the anemia is due to deficiency of vitamin B12 and folic acid The macrocytes in this condition is usually “oval” - hence they are also called as MACRO OVALOCYTES

NON MEGALOBLASTIC MACROCYTIC ANEMIAS These are disorders in which the macrocytosis is not due to vitamin B12 or folic acid deficiency Here the macrocytes are “ROUND” The conditions in which such round macrocytes are seen are Reticulocytosis Hypothyroidism / myxedema Myelodysplastic syndrome Scurvy (Vit-C dif) Sideroblastic anemia Liver disorders

MEGALOBLASTIC ANEMIA Vitamin B12 and folic acid are important nutrients required in the process of nuclear maturation They are required during erythropoiesis (during DNA synthesis) These anemias may be caused because of a nutritional deficiency or impaired absorption mainly.

MEGALOBLASTIC ANEMIA Impaired DNA synthesis leading to defective cell maturation and cell division Nuclear maturation delays from the cytoplasmic maturation – NUCLEAR CYTOPLASMIC ASYNCHRONY Abnormally large erythroid precursors and red cells

Folic Acid: Daily requirement: Transportation: It a vitamin which is yellow in colour, water soluble, necessary for the production of the RBC, WBC and platelets. It is not synthesized in the body. It is found in large number of green fresh vegetables, fruits. Daily requirement: The human body needs about 100-150 µg daily. Absorption: It is absorbed in the Duodenum and Jejunum. Transportation: Weakly bound to albumin.

METABOLIC FUNCTION Purine synthesis Conversion of homocysteine to methionine ( which also requires B12 )

FOLIC ACID DEFICIENCY INCREASED DEMAND DECREASED INTAKE DECREASED ABSORPTION METABOLIC INHIBITION

INCREASED DEMAND Pregnancy Lactation Infancy Puberty and growth period Patients with chronic hemolytic anemias Disseminated cancer

DECREASED INTAKE Elderly Lower socio economic status Chronic alcoholics

DECREASED ABSORPTION Acidic food substances in foods like legumes, beans Drugs like phenytoin, oral contraceptives Celiac disease which affect the gut absorption Heat sensitive – more loss during cooking

METABOLIC INHIBITION

Vitamin B12: This vitamin is synthesized in nature by micro-organism in the intestine of man and animals, but we can not obtain it from the bacteria in our bodies, because it is synthesizing in the large colon after the site of absorption and it is wasted in the faeces in about 5µg/day. So we obtain it from animal food such as liver, kidney, meat and dairy products as milk and cheese.

VITAMIN B12 Abundant in animal foods Microorganisms are the ultimate origin of cobalamin It is stored in liver for many years It is efficiently reabsorbed from bile It is resistant to cooking and boiling

Diary requirements: The human body needs about 1-2 µg daily. Absorption: B12 is combined with glycoprotein called the intrinsic factor (IF), which is synthesized in the gastric cells. The absorption occurs in the distal ileum. Transportation: Transport by a protein synthesized in the liver called Transcobalamine II, which carry vitamin B12 to liver, nerves and bone marrow.

VITAMIN B12 DEFICIENCY INCREASED REQUIREMENT DECREASED INTAKE IMPAIRED ABSORPTION

INCREASED DEMAND Pregnancy Lactation Puberty Growth period Hyperthyroidism Disseminated cancer

DECREASED INTAKE Inadequate intake Vegetarian diet

IMPAIRED ABSORPTION INTRINSIC FACTOR DEFICIENCY due to chronic gastritis or antibodies against stomach cells. - PERNICIOUS ANEMIA - GASTRECTOMY Malabsorption states Diffuse intestinal diseases. Eg., lymphoma, systemic sclerosis Competitive parasitic uptake – fish tapeworm Bacterial overgrowth

Pathophysiology Folates are compounds derived from folic acid that are involved in numerous metabolic reactions Generally folates act as donor of single carbon groups dUMP=deoxyuridylate monophosphate DTMP=deoxythymidylate monophosphate One such reaction is thymidylate synthesis Defective thymidylate synthesis leads to defective DNA synthesis Megaloblastic anemia

B12 and folate B12 is a known cofactor in 2 enzymatic reactions (1) B12 is a cofactor of methionine synthase which is required to regenerate folate within the cell

Other sequelae of B12 deficiency (2) Conversion of methylmalonyl CoA and finally to succinyl-CoA Co factor for methylmalonyl CoA Failure of this pathway leads to abnormalities in neuronal membrane synthesis. Also, methionine is a precursor for S-adenosylmethionine-a metabolite critical for normal function of the nervous system. Neurologic defects seen with B12 deficiency Peripheral neuropathy Disturbances of vibratory sense and proprioception Dorsal and lateral column demyelination Spastic ataxia Brain Dementia, psychosis, somnolence Although anemia may respond to folate therapy, neurologic findings will not Neurologic findings may be permanent if not treated early 1 Up-to-Date

Folate deficiency Dietary sources Metabolism Present in animal and vegetable products Asparagus, broccoli, spinach, lettuce, lima beans (>1mg/100g dry weight) Liver, yeast, mushrooms, oranges Cooking depletes food of folate Metabolism Absorbed most actively in the jejunum and upper ileum Body stores are 5-10 mg (liver) Minimal daily intake is 50 micrograms Higher for pregnancy and lactation Folate deficiency can lead to birth defects (neural tube defects) If intake is reduced to 5 micrograms/day, megaloblastic anemia will develop in ~4 months

Folate deficiency Causes of folate deficiency Decreased intake (most common) Dietary deficiency Infants on a Goat’s milk diet Small intestinal disease Tropical sprue Celiac sprue (gluten sensitive enteropathy) Increased requirement Pregnancy Alcoholism Hemolytic anemia Leukemia

B12 deficiency Dietary sources Metabolism Animal products (meat and dairy) Metabolism Ingested B12 is protein bound Trypsin and acid in stomach release B12 B12 binds R-binding protein which carries it to the jejunum Also in the stomach (fundus and body) intrinsic factor is secreted. In the jejunum, pepsin releases B12 from R-binding protein B12 binds intrinsic factor and is carried to the ileum B12 is absorbed in the ileum Body stores 2-5mg (mostly in the liver) Need 3-5 micrograms per day for maintenance of stores Increased need in pregnancy, lactation, growth Depletion takes longer that folate It takes years to develop megaloblastic anemia due to B12 deficiency

B12 deficiency Decreased intake Impaired absorption Veganism Gastric Poor stomach acidity Gastrectomy Pernicious anemia Decreased secretion of intrinsic factor due to gastric atrophy Chronic pancreatitis Decreased digestive enzyme secretion Intestinal disease Ileal resection Ileal disease Chron’s disease Celiac sprue Fish tapeworm Diphyllobothrium latum Blind loop Intestinal bacterial overgrowth

Diagnosis Clinical findings Morphology-peripheral blood and marrow Megaloblastoid morphologic changes Folate deficiency Folate, serum level Reflects recent levels of ingestion Falsely increased with hemolysis RBC Folate Reflects stores (2-3 months) Will be decreased in B12 deficiency B12 deficiency Folate levels are decreased in B12 deficiency, should check both in tandem B12, serum level Serum methylmalonic acid and homocystine levels may be more sensitive B12 deficiency, both are elevated sensitivity 94%, specificity 99% Folate deficiency, only homocystine levels are elevated May miss 10-26% of patients with serum B12 levels alone Schillings test Helps to identify the source of B12 deficiency Homocystine MMA

Sequence of changes in megaloblastic anemia Vitamin levels decrease Neutrophil hypersegmentation Oval macrocytosis in the peripheral blood Megaloblastic changes in the marrow Anemia

MEGALOPLASTIC ANAEMIA. Affect all marrow elements. Neurologic symptoms (dorsal columns) Ineffective erythropoiesis: High indirect bilirubin Very high LDH

Ineffective hematopoiesis Because of the faulty megaloblastic maturation seen in the marrow, cells die during cell division Hypercellular marrow due to erythroid hyperplasia with increased red blood cell precursors in the marrow but a macrocytic anemia in the periphery Release of cell constituents Increased bilirubin Increased LDH

CLINICAL FEATURES Patients develop all general symptoms and signs of the anaemia. Knuckle pigmentation Angular stomatitis Atrophic glossitis- “beefy” tongue Neurological disorders: sever deficiency of the folic acid causes neuropathies diseases. Deficiency during pregnancy causes neural tube defect.

Diagnostic algorithm

Schillings test Allows one to determine the level at which B12 deficiency is occurring Dietary deficiency Malabsorption Absence of intrinsic factor

Screening hematology laboratory tests for megaloblastic anemia CBC Macrocytic anemia MCV is usually >110 fL and often >120 fL Pancytopenia is seen in some cases Peripheral blood morphology Oval macrocytes Nucleated RBCs, Howell Jolly bodies, basophilic stippling, Cabot rings Neutrophil nuclear hypersegmentation five 5 lobed neutrophils per 100 WBC One six lobed neutrophil

Megaloblastic anemia: Morphology--Blood Megaloblastoid Normal Peripheral blood Macro-ovalocytes Neutrophil nuclear hypersegmentation Very sensitive and specific

Howell Jolly body. - Round, dark, refractile RBC inclusion Howell Jolly body - Round, dark, refractile RBC inclusion - Nuclear remnant - Most commonly seen in hyposplenism Basophilic stippling - Multiple small blue dots dispersed regularly throughout the RBC - RNA complexes Cabot ring - Ring shaped, thin - May be figure of 8 shaped - Microtubule, remnants of of the mitotic spindle - Rarely seen

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 sever.

PERIPHERAL SMEAR RBC: Poikilocytosis - tear drops and schistocytes Anisocytosis - oval macrocytes -Macro ovalocytes (macrocytic normochromic) -well hemogloibised, thicker than normal -inclusions like HOWELL JOLLY BODIES, basophilic stippling, Cabot rings

PERIPHERAL SMEAR WBC: Normal count or reduced count Hypersegmented neutrophils (>5 lobes) MACRO POLYMORPHO NUCLEAR CELLS (Macropolys) PLATELETS: Normal or decreased

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

MEGALOBLAST Abnormally large precursor Deeply basophilic royal blue cytoplasm Fine chromatin with prominent nucleoli Nuclear cytoplasmic asynchrony Abnormal mitoses Maturation arrest

Megaloblastic anemias Morphology Bone marrow Nuclear cytoplasmic asynchrony in both erythroid and myeloid lineages Megaloblastic Normal Megaloblastic Megaloblastic

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

TESTS FOR FOLATE AND B12 DEFICIENCY Serum folate assay Red cell folate assay Serum B12 assay

Pernicious Anemia Decreased secretion of intrinsic factor due to gastric atrophy and loss of parietal cells More common in individuals of Northern European descent greater than age 50 Most common cause of vitamin B12 deficiency Diagnosis Intrinsic factor antibodies (commonly blocks B12 binding site) Sensitivity 50-84% Specificity ~100% Parietal cell antibodies Less specific ~50% sensitive

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 B12 and IF binding Type II antibody – prevents binding of IF-B12 complex with ileal receptors Type III antibody – 85-90% patients – against specific structures in the parietal cell Associated with other autoimmune diseases like autoimmune thyroiditis

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 B12 Elevated levels of homocysteine Striking reticulocytosis after parenteral administration of vitamin B12 Serum antibodies to intrinsic factor

Therapy B12 Oral or intramuscular therapy Both work well but IM is preferred for patients with an absorption problem Folic acid 1-5mg orally for one to four months (or till macrocytic anemia resolves) Folic acid will correct the hematologic but not the neurologic sequelae of B12 deficiency !!!!!MUST rule out B12 deficiency Response Reticulocytes rise after Day 4 and peak on day 5-8 Hemoglobin rises 2-3g/dL every 2 weeks Marrow begins to respond within days Hypersegmented neutrophils may persist for up to 2 weeks

Non-megaloblastic macrocytic anemias Alcoholism Liver disease Hemolysis, acute bleed Hypothyroidism Aplastic anemia Artifact RBC clumping cold agglutinin disease Hyperglycemia swelling

Alcohol Common cause of macrocytosis Regular ingestion of 80g alcohol per day (1 bottle of wine) Abstinence form alcohol leads to resolution of macrocytosis in 2-4 months ~90% of alcoholics have a macroytosis (100-110 fL) before anemia develops Multifactorial in etiology Direct toxic effect Associated liver disease Reticulocytosis related to GI bleeding Co-incident folate deficiency

Liver disease Hyperlipidemia Hypothyroidism Artifact Macrocytosis and target cells Mechanism not well understood May be related to increased lipid deposition on RBC membranes Hyperlipidemia Hypothyroidism Mechanism unknown ~10% of patients with autoimmune thyroiditis have pernicious anemia Artifact Hyperglycemia (>600) RBC agglutination

Summary Macrocytic anemias can be megaloblastic or non megaloblastic Megaloblastic anemia has characteristic morphologic features Nuclear cytoplasmic asynchrony B12 and folate deficiency are 2 reversible causes of megaloblastic anemia

Case A 66 yo homeless man presents with generalized confusion, bruises and lacerations secondary to a witnessed fall down a flight of steps Physical exam revealed a laparotomy scar A CBC shows the following 7.8 2. 108 24 MCV 129 A peripheral blood smear is reviewed What additional laboratory tests would you like to request? http://path.upmc.edu/cases/case428.html

Diagnosis ?? ETOH Elevated B12 63 pg/mL (211 – 911) Folate 13.2 ng/mL (>5.4) RBC Folate 805 ng/mL (293 – 809) Diagnosis ?? Megaloblastic anemia B12 deficiency As the patient’s mental status cleared he reported that the scar on his abdomen was secondary to a partial bowel resection including a large segment of ileum

Case RPI=3.4 Reticulocyte 9% What is the RPI? [9% X (30/45)]/1.75 12 year old boy with no significant past medical history presents to his pediatrician with persistent poor appetite and weakness 1 week following an acute viral illness He is found to be anemic Reticulocyte 9% What is the RPI? 10 [retic% X (Pt Hct/45)]/MAT MAT = 1 + 0.05(45 - Pt's HCT) ) (1.75 in our case) 5 180 30 [9% X (30/45)]/1.75 MCV 91 MCHC 35.9 RPI=3.4

Additional labs and peripheral blood smear Haptoglobin Indirect bilirubin No increase in: Plasma hemoglobin Urine hemoglobin Extravascular hemolysis Splenomegaly on exam Of note, the mother had a similar peripheral blood smear