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

2. 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

3. 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

4. Big Issue‼
MCV=mean corpuscular volume

5. 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

6. 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

7. 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

8. 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

9. 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

10. 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 * * *

11. 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

12. 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

13. 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.

14. 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

15. 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 µg daily. Absorption:
It is absorbed in the Duodenum and Jejunum.
Transportation:
Weakly bound to albumin.

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

18. FOLIC ACID DEFICIENCY INCREASED DEMAND DECREASED INTAKE
DECREASED ABSORPTION
METABOLIC INHIBITION

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

20. DECREASED INTAKE Elderly Lower socio economic status
Chronic alcoholics

21. 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

22. METABOLIC INHIBITION

23. 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.

24. 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

25. 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.

26. VITAMIN B12 DEFICIENCY INCREASED REQUIREMENT DECREASED INTAKE
IMPAIRED ABSORPTION

27. INCREASED DEMAND Pregnancy Lactation Puberty Growth period
Hyperthyroidism
Disseminated cancer

28. DECREASED INTAKE
Inadequate intake
Vegetarian diet

29. 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

30. 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

31. 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

32. 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

33. 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

34. 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

35. 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

36. 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

37. 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

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

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

40. 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

41. 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.

42. Diagnostic algorithm

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

44. 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

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

46. 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

47. 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.

48. 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

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

55. 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

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

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

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

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

64. 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

65. 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

66. 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

67. 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

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

69. 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 ( fL) before anemia develops
Multifactorial in etiology
Direct toxic effect
Associated liver disease
Reticulocytosis related to GI bleeding
Co-incident folate deficiency

70. 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

71. 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

72. 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?

73. Diagnosis ?? ETOH Elevated B12 63 pg/mL (211 – 911)
Folate 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

74. 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 = (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

75. 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