Macrocytic Anemia.pptx

Approch for macrocytic/megaloblastic Anemia

1. APPROACH TO DIAGNOSIS A CASE OF MACROCYTIC ANAEMIA BY DR.RUSHANG DAVE UNDER THE GUIDEINENCE O F DR . GARIMA ANANDANI

2. SYNOPSIS Introduction Macrocytosis without anemia Factitious macrocytosis Classification of macrocytic anemia Megaloblastic anemia – Causes Biochemical basis, folate and B12 metabolism Laboratory diagnosis Treatment and response assessment Non-megaloblastic anemia Causes and morphologic findings. Summary

3. Morphologic classification RBC indices MCV < 80fL Microcytic anemia MCV 80 to 100fL Normocytic anemia MCV > 100fL Macrocytic anemia

4. Definition These are the anaemias in which the RBC have an MCV of greater than 100 fl. There are two groups of macrocytic anaemias - Megaloblastic anemia - Non megaloblastic macrocytic anemia

5. Macrocytosis Without Anemia Neonatal RBCs (at birth 100-130 fL) Genetic predisposition Early cases of vitamin B12 deficiency. (Macrocytosis characteristically precedes the development of anemia and may even do so by several years) Hence raise in MCV should not be ignored and correlated clinically

6. FACTITIOUS MACROCYTOSIS

7. Case 1 45 year old female Hb of 7.1 g/dL RBC 0.8 million/cumm MCV – 111.9 fL MCH – 86.4 pg MCHC – 77.8 g/dL Agglutination of RBCs result in grossly abnormal indices RBC count, MCV, MCHC

8. Case 2 32 yr female with menorrhagia Hb- 8.8 g/dL RBC – 2.7 million MCV – 101 fL MCH – 27.6 pg MCHC – 31.8 g/dL Polychromatophils 20% larger in size As a very rough rule of thumb, for every 1% increase in the reticulocyte percentage, there will be an increase of 1 fL in the MCV.

9. Case 3 54 yr male Hb - 9.8 g/ dL RBC – 3.5 million MCV – 109 fL MCH – 27.6 pg MCHC – 28.8 g/ dL HbA1c- 9% RBCs are glucose loaded and swell when they are mixed with isotonic diluent MCV is elevated, PCV is increased and MCHC is abnormally low.

10. Case 4 EDTA induced changes Increase in red cell size with time, due to degenerative changes that permits ingress of water into the cells artefactual increase in RBC size could mask microcytosis and lead to erroneous diagnoses of macrocytosis . Other morphological changes such as spherocytosis , echinocytosis & spheroechinocytosis were observed from Day 2 onwards

11. Factitious macrocytosis Hyperosmolar conditions Uncontrolled diabetes where RBCs are glucose loaded and swell when they are mixed with isotonic diluent MCV is elevated, PCV is increased and MCHC is abnormally low . Prolonged storage especially in EDTA Extreme leucocytosis – leucocytes may enter RBC channel Methanol poisoning – increasing MCV value.

12. CAUSES OF MACROCYTIC ANEMIA Macrocytic anemia Megaloblastic Non megaloblastic

13. Megaloblast The term ‘megaloblast’ is a designation that was first applied by Ehrlich to the abnormal erythrocyte precursors found in the bone marrow of patients with pernicious anemia Megaloblasts are characterized by their large size and by specific alterations in the appearance of their nuclear chromatin – Sieve like, nucleo-cytoplasmic asynchrony Normoblastic erythroid precursor Megaloblastic erythroid precursor

14. Causes Folate deficiency Vitamin B12 deficiency Drugs Inherited disorders Orotic aciduria Thiamine responsive megaloblastic anemia Dihydrofolate reductase deficiency Transcobalamin II deficiency Homocysteinuria and methyl malonic aciduria Lesch-Nyhan syndrome

15. Biochemical basis of megaloblast

16. B12 and folate metabolism

17. B12 deficiency Methyl malonyl coA Succinyl coA Methyl malonic acid Myelin destabilizer Deoxyadenosyl B12

18. Vitamin B12 (Cobalamin) Present in animal protein predominantly. Stored in liver Co factor for one carbon metabolism RDA is 2.4 μg in adults The total amount of vitamin B 12 stored in body is about 2–5 mg in adults. 2 important forms in body are methyl cobalamin and deoxyadenosyl cobalamin

19. Mechanism of absorption

20. Causes of B12 deficiency Dietary deficiency (Vegans)  Lack of intrinsic factor   Pernicious anemia  Gastric surgery – Bariatric surgery   Prolonged use of PPI   Biologic competition for vitamin B 12   Small bowel bacterial overgrowth Fish tapeworm disease  Familial selective vitamin B 12 malabsorption  Chronic pancreatic disease  Zollinger-Ellison syndrome  Diseases of the ileum Ileal resection  Regional enteritis/ intestinal TB

21. Clinical features Malaise, irritability, nausea , vomiting, abdominal pain, diarrhea, anorexia, cognitive impairment, forgetfulness. Mild jaundice Atrophy of tongue papillae gives rise to a beefy red tongue. Aphthous stomatitis and oral soreness in some patients even without anemia. Transient darkening of nails and skin (knuckle hyperpigmentation) Beefy red tongue Aphthous ulcer

22. Neurological manifestations Neurologic changes can precede anemia General symmetricalness of manifestations is typical The earliest manifestations are loss of vibratory sense in the feet and numbness, tingling, and loss of fine sensation Posterior and lateral column degeneration Ataxia, spasticity, gait disturbances , impotence, and loss of bladder and bowel control, gait disturbances, neuropsychiatric manifestations are common signs of advanced involvement

23. Folate RDA - 400 μg daily for adults 600 μg for pregnant women 500 μg for lactating women Food folate, mostly methylTHF and formylTHF, is labile Storage of Folate – upto 7.5 mg in liver, depleted within 100 days

24. Causes of folate deficiency   Dietary deficiency Decreased absorption (Jejunal resection, Celiac disease)  Increased requirements Pregnancy Infancy Chronic hemolytic anemia, myeloproliferative conditions   Alcoholism Drug-induced folate deficiency – Anti convulsants

25. PERNICIOUS ANAEMIA Immunological mediated-Gastric mucosa Chronic atrophic gastritis-loss of parietal cells, infiltrate by lymphocytes and plasma cells, megaloblastic changes of mucosal cells. Three types of antibodies Type I- 75%- Ab that blocks binding of B12 to IF- plasma and gastric juice Type II- Ab blocks binding of IF-B12 complex to Ileal receptor. Type III- Ab 85%-recognize α and β subunit of gastric proton pump.

26. Lab parameters Hb values <7 or 8 g/dl are not unusual The MCV usually between 110 and 130 fl RDW is substantially increased , and the increase may precede the development of anemia Reticulocyte count is reduced due to ineffective erythropoiesis As megaloblastic anemia progresses, neutropenia and thrombocytopenia develop The pancytopenia may mimic aplastic anemia Serum lactate dehydrogenase and indirect bilirubin levels – elevated reflecting the intramedullary destruction of RBCs as well as shortened life span

27. PS The main products of megaloblastic erythropoiesis are oval macrocytes with reduced or absent central pallor. Hypersegmentation ( 5% with 5 lobes or 1 with 6 lobes) is among the first hematologic abnormalities to appear It persists for an average of 14 days after institution of specific therapy

28. Macrocytic anemia: CBC report from a case of macrocytic anemia shows increased MCV (111.7 /L) and MCH (37.7 pg). However MCHC is 33.7 g/ dL in the normal range. RDW as increased to 30.3%. Histogram curve (green) is shitted to right suggesting increased MCV. The green curve is widened indicating moderate degree of anisopoikilocytosis and therefore RDW-CV is increased to 30.3%. Red curve is of normocytic normochromic red cells (Normal}

29. Bone marrow examination The initial marrow change is megaloblastosis , even before macrocytosis appears in the peripheral blood. Bone marrow is markedly hypercellular with erythroid hyperplasia Thus reversal of M : E ratio to even 1 : 8.

30. The hallmark in the marrow is the nuclear- cytoplasmic maturation dissociation which is best seen in the erythroid precursors resulting in megaloblastosis Megaloblasts have open sieve like nuclear chromatin and basophilic cytoplasm.

31. There is preponderance of early megaloblasts which are more than intermediate and late megaloblasts Intermediate and late megaloblast die in the marrow because of unbalanced, nuclear and cytoplasmic maturation. There is widespread intra- medullary apoptosis of defective erythroid cell precursors resulting in rise of unconjugated bilirubin .

32. Though marrow is very hypercellular and full of megaloblasts , only 10—20% of the potential progeny of proerythroblasts survive to become the macrocytic red cells and this ineffective erythropoiesis results in anemia. Howell Jolly bodies and stippling of cytoplasm is seen in some megaloblasts . Both abnormal and normal mitoses are observed frequently . Nuclei of megaloblasts demonstrate features of dyserythropoiesis like nuclear budding, irregular nuclei, dumb-bell shaped nuclei, and nuclear fragments etc ,

33. D) Marrow aspirate shows a giant megaloblast {*) (at the bottom of the picture)with megaloblasts showing features of dyserythropoiesis in the form of irregular nuclei . E) Bone marrow shows erythroid hyperplasia. Mitosis is frequent .

34. There is abnormal hemoglobinization of cytoplasm of megaloblasts ., Megaloblastosis changes to normoblastic erythropoiesis within 12 hours of giving Vit . B12/FA and marrow morphology reverts to normal within 2-3 days. Therefore, bone marrow should be carried out pretreatment if required.

35. The misshapen neutrophils fray and the cells disintegrate resulting in neutropenia — ineffective myelopoiesis , Giant metamyelocytes show bizzare shapes of the nuclei (misshapen nuclei), and abnormal chromatin and staining character

36. Megakaryopoiesis Is less disturbed as compared to erythropoiesis and myelopoiesis , Megakaryocytes of variable maturity display nuclei with open chromatin pattern and complex nuclear lobular hypersegmentation . Megakaryocytes show larger size, fragmentation and very few platelets are formed— some of them being megathrombocytes . This "ineffective thrombopoiesis accounts for reduced platelet count.

37. Trephine biopsy Bone marrow trephine biopsy displays preponderance of early megaloblasts with uniform size and shape, having open nuclear chromatin with linear 1-4 nucleoli and frequent mitoses . It is essential to differentiate these cells from blast cells which are seen as small clusters (ALIP-Abnormal localization of miniature precursors) in MDS. Morphologic aberrations are also apparent in myeloid cells and‘giant metamyelocytes and band forms' are pathognomonic of megaloblastosis ,

38. Bone marrow trephine biopsy shows eryihroid hyperplasia with preponderance of early magaloblasts which are of uniform size with delicate nuclear membrane, vesicular nuclei and 1—3 linear nucleoli which touch the nuclear membrane,

39. Vitamin B12 Levels Standard initial routine diagnostic test Microbiological assay replaced by automated radioimmunoassay, chemiluminescence In cobalamin deficiency, serum cobalamin levels fall below the generally accepted cutpoint of 200 to 250 ng/L ( <125 pmol/L ) Measures total Cobalamin ( Tc I, Tc II, Tc III) – hence may be elevated in CML

40. Methyl Malonic acid Increases in serum and urine in cobalamin deficiency but not folate deficiency Serum levels are elevated in 98.4% of patients with symptomatic cobalamin deficiency Levels often exceed >0.75 µmol/l in symptomatic cobalamin deficiency but are usually only mildly elevated in subclinical deficiency Falsely elevated in hemoconcentration, renal disease, small bowel bacterial overgrowth MMA is a more specific test for cobalamin deficiency than homocysteine

41. Total homocysteine Homocysteine rises with either folate or cobalamin deficiency A normal result helps to exclude clinically relevant deficiency Levels above 12 to 14 μmol/L in women and 14 to 15 μmol/L in men are generally regarded as elevated. Homocysteine rises in many acquired and genetic conditions Folate intake and serum creatinine levels are major determinants of homocysteine status

42. Holotranscobalamin Holotranscobalamin (HoloTC) – measures TC II, the ‘active’ fraction of plasma cobalamin Relatively new test Immunoassay for this fraction is now available Values for HoloTC in healthy individuals are 35–171 pmol/l Better sensitivity and specificity characteristics than B12 levels

43. Deoxyuridine suppression test Abnormal Deoxyuridine suppression serves as a sensitive index of the presence of megaloblastosis due to vitamin B12 or folate deficiency. Test reflects impaired conversion of deoxyuridylate to thymidilate done for research purposes Can be done even in subclinical deficiency

44. Cobalamin (156-672 pmol/L) >250 pmol/L No deficiency <125 pmol/L B12 deficiency 125-250 pmol/L Grey area 0.29-0.75 µmol/L Grey area (Rpt after 1 yr) <0.29 µmol/L No deficiency >0.75 µmol/L B12 deficiency MMA(0.08-0.28 µmol/L)

45. TEST TO DIAGNOSIS FOR B12 DEFICIENCY: The Schilling test is a 3 stage test. Stage 1 - Give radioactive labeled vitamin B12 orally. Then screen the patient's urine for the radio labeled B12. *If the radioactive B12 appears in the urine, the patient has successfully absorbed the B12 and the abnormality is a dietary deficiency of Vitamin B12. End of test. *If no or scant B12 appears in the urine, then the ingested B12 was not absorbed. The defect in absorption is not due to dietary deficiency. Proceed to Stage 2.

46. Stage 2 - Give oral radio labeled B12 with oral intrinsic factor and again screen the patient's urine for B12. *If B12 is found in the urine, then the defect is due to a lack of intrinsic factor . End of test. *If no or scant B12 is present in the urine, then the defect is not due to a lack of intrinsic factor (because absorption is not corrected by giving extrinsic IF). The defect is at the intestinal level. Proceed to stage 3 SCHILLING TEST (Contd..)

47. Stage 3 - Treat patient with 7-10 days of antibiotics . Then give radio labeled B12. *If decreased Ileal absorption is due to bacterial overgrowth, antibiotics will correct this absorptive defect and B12 will be detected in the urine. *If decreased Ileal absorption is secondary to an anatomic defect , antibiotics will have no effect on B12 absorption and no B12 will be found in the urine. SCHILLING TEST (Contd..)

48. Tests to diagnose cause of B12 deficiency Nothing further if diet, resection history, very elderly patients Anti IF antibodies – 100% specific, 70% sensitive for Pernicious anemia All patients with anaemia, neuropathy or glossitis, and suspected of having pernicious anaemia, should be tested for anti-IFAB regardless of cobalamin levels (Grade 1A) Gastric anti parietal cell antibodies – May be +ve, very low specificity

49. Other tests Cobasorb – Functional measure of Vit B12 absorption Baseline holoTC measured Oral B12 given Increased holoTC – normal absorption Unchanged HoloTC – No absorption Needs further evaluation before introduction into routine clinical practice Endoscopy for gastric malignancy every 5 years in case of atrophic gastritis

50. Tests to diagnose Folate deficiency Serum Folate The definition of folate deficiency in patients with megaloblastic anemia, is a serum folate level below 3 μg/L. Fluctuates with folate intake, falsely elevated in hemolysis RBC folate MethylTHF (60%) and formylTHF (26%) are the major folates in red cells. Red cell folate content is constant and is viewed as a truer indicator of tissue folate Red cell levels below 160 μg/L are usually considered low

51. Investigation for folate FIGLU test Histidine ----------------- → FIGLU ------------------------- → Glutamate ↑ THF Oral challenge of Histidine Increased urinary excretion of FIGLU in folate deficiency.

52. Gastric biopsy : Cases with, pernicious anemia demonstrate gastric atrophy with loss of gastric glands and parietal cells. There is infiltration of lamina propria by lymphocytes and plasma cells.

53. Indications for bone marrow Generally no role except Lack of facilities for serum B12 and folate levels Atypical clinical presentation Splenomegaly Bleeding Sternal tenderness Presence of circulating blasts

54. Treatment Treatment with folate alone in B12 deficiency cases may result in masking of peripheral blood picture and neurological symptoms may continue worsening . Standard initial therapy for patients without neurological involvement is 1000 µg hydroxocobalamin intramuscularly (I.M) three times a week for 2 weeks Patients with neurological symptoms should receive 1000 µg hydroxocobalamin I.M. on alternate days until there is no further improvement Folate is also empirically administered 400μg to 1mg/day Folate deficiency : 5 mg of folic acid daily is taken for 4 months .

55. Response to treatment Often response to empirical treatment is used as a diagnostic test Reticulocyte level starts by 2-3 days and peaks by 6-8 days Marrow is completely normoblastic after 3-4 days. Giant metamyelocytes persists for 12 days A complete blood cell count 10-14 days after starting the treatment should reveal a rise in hemoglobin (0.1g/dl per day) and a decrease in MCV F ull hematologic response should occur within 8 weeks.

56. Masked megaloblastosis Deficiency of vitamins without classical blood picture and bone marrow picture. Coexisting Iron deficiency or thalassemia PS findings : Dimorphic population of macroovalocytes and microcytic hypochromic RBCs Increased RDW MCV may be normal Hypochromic ovalocytes is a clue Megaloblastic anemia (crisis) may be the initial presentation in congenital hemolytic anemia

57. Non-megaloblastic macrocytic anaemia Represent macrocytic anemias in which the RBC precursors appear normal without the characteristic nuclear and cytoplasmic findings of megaloblasts DNA synthesis is unimpaired . Macrocytosis is usually mild; the MCV usually ranges from 100 to 110 fl and rarely exceeds 120 fL

58. Causes Aplastic anemia Myelodysplastic syndrome Acquired sideroblastic anemia Congenital dyserythropoietic anemia types I and III Diamond-Blackfan anemia Erythroleukemia Alcoholism Liver disease Hypothyroidism Copper deficiency HIV Drugs – (independent of DNA synthesis ) Valproic acid, Recombinant EPO, Sulfonamides

59. Case 5 55 male with ascites, splenomegaly Hb 8.5 g/dl TLC 3800/cmm Platelet 1,25,000/cmm N 70, L 26, E2, M2 MCV 103 fl MCH 28 pg MCHC 29 gm/dl Retic 2.5%

60. Alcoholism and liver disease Associated with thin macrocytes, defined as cells with increased surface area but without a corresponding increase in volume Consequence of excessive membrane lipids, especially cholesterol , but also phospholipids. The MCV rarely exceeds 115 fl Cause Faulty lipid metabolism Associated Folate deficiency Alcoholics may be associated with spur-cell hemolytic anemia Poorly defined direct effects of alcohol on the bone marrow Serum and erythrocyte folate levels are usually normal , and the macrocytosis does not respond to folate treatment – abstaining from alcohol brings down the MCV

61. Case 6 16 year male Severe anemia and bleeding gums – 2 months Hb 4 gm/dl TLC 1600/cmm N 10 L 90 Platelet count 20,000/cmm Reticulocyte 0.1% MCV 108 fl 10-20 % aplastic anemia have macrocytosis less than 110 fl Much severe pancytopenia No oval macrocytes or hypersegmented neutrophils Lymphocytic preponderance

62. Case 7 4 month old child Severe pallor and supernumerary thumb Hb 5 gm/dl TLC 5600/cmm Platelet 2,15,000/cmm Retic < 0.2% Congenital red cell hypoplasia or Diamond-Blackfan anemia is a pure RBC aplasia in children. Congenital abnormalities, including short stature and skeletal defects, specially of thumbs The diagnosis of DBA is suggested by a macrocytic (although sometimes normocytic) anemia with reticulocytopenia presenting in the first 6 months of life. Bone marrow examination reveals decreased erythroid precursors.

63. Case 8 14 year old male Refractory anemia since early childhood Multiple transfusions Hb 6 gm/dl MCV 110 fl TLC 8700/cmm Platelet 3,50,000/cmm DLC – normal Retic 1% Peripheral blood in CDA-I Bone marrow in CDA-I

64. Congenital dyserythopoietic anemia CDA - characterized by ineffective erythropoiesis and dyserythropoiesis. Morphologically abnormal erythroblasts in the bone marrow with multinuclearity , nuclear bridges, karyorrhexis, or megaloblastic changes. Morphologically , the anemia is generally mildly macrocytic in CDA types I and III and is normocytic in CDA type II. Bone marrow in CDA-III Polychromatophilic erythroblasts with multinucleation

65. Case 9 70 year male Progressive pallor Hb 6 gm/dl TLC 2900/cmm Platelet 45,000/cmm

66. Myelodysplastic syndrome Erythrocyte morphology is usually macrocytic Oval macrocytosis is the best-recognized erythrocyte abnormality. In extreme cases, elliptocytes , teardrops, schistocytes, stomatocytes, or acanthocytes Basophilic stippling, Howell-Jolly bodies, and megaloblastoid nucleated red cells Circulating blasts 5 q- syndrome – classically has macrocytosis Dyspoietic m egaloblastic morphology is also a feature of Erythroleukemia

67. CASE 10 A 69-year-old man presented with a 2-year history of progressive thrombocytosis , referred to exclude essential thrombocythemia . no palpable splenomegaly . Hemoglobin- 10.9 g/dl; mean corpuscular volume 100 fL ; platelets 9.3lacs; The peripheral blood smear showed marked thrombocytosis , giant platelets, and red cell dysplastic changes including oval cells, target cells, numerous red cell stippling, and Pappenheimer bodies (panel A). These findings suggested acquired sideroblastic anemia and an associated myeloproliferative neoplasm. 

68. Case 11 A 57-year-old African American woman with shortness of breath and chest pain complete blood count profile showed  pancytopenia , white blood cell count of 3.3 × 10 3 / μL , a hemoglobin level of 4.4 g/ dL , platelet count of 29000/ cumm Numerous nucleated red blood cells, erythroblasts, and a rare circulating  megakaryoblast were identified on peripheral smear examination.

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