Inherited transcobalamin II deficiency (Autosomal recessive, treated with large doses of Vitamin B12, to push it into the cell- 1000microgm- IM- Weekly- lifelong)
Imerslund – Grasbeck disease (Autosomal recessive condition, mutation in cubulin receptor gene, Associated with proteinuria)
Nitrous oxide anaesthetic inhalation (Destruction of endogenous cobalamin by oxidation, resulting in acute megaloblastic anemia, which presents with rapidly developing thrombocytopenia and leukopenia)
Causes of Folic acid deficiency
Decreased intake
Inadequate content in diet (Folate is destroyed by cooking)
Neurological manifestations- Occur due to cobalamin deficiency (Sometimes occurs without anemia or macrocytosis but BM shows megaloblastic changes. These manifestations get aggravated with administration of folic acid without vitamin B12 supplementation)
Peripheral neuropathy: Paraesthesia, muscle weakness difficultly in walking, Burning sensation of tongue, Vague abdominal pain
Demyelination of posterior & pyramidal tracts of spinal cord (Subacute combined degeneration)- Loss of vibration sense for 256HZ but not for 128Hz, Extensor plantars, Spastic paraplegia, sensory ataxia, severe paraesthesia in lower limbs, Sphincter disturbance, Decreased reflexes, Romberg’s and Babinski’s sign- Positive, Loss of position sense in second toe
Optic atrophy & retro bulbar neuritis- Seen especially in patient who smoke.
Nonhematological effects of folate deficiency
Arterial thrombosis due to increased homocysteine (This role is uncertain)
Syndrome similar to HELLP- Differentiated by presence of anemia and macrocytosis
Colon cancer- Supplementation of folate decreases risk of colon cancer by 31% (Folate helps in DNA repair. But if already tumour cells are formed, folate helps in their proliferation)
Investigations:
Hemogram:
Hemoglobin –It is less than 12gm/dl, only in 29% cases.
MCV-Elevated to more than 100fL
MCH-Elevated
MCHC- Normal
Macrocytic anemia, ovalocytosis (Co-existing iron deficiency/ thalassemia/ inflammation may prevent macrocytosis)
Poikilocytosis if anemia is severe
Normo / hyperchromasia
Increased polychromatophilic cells
Many nucleated RBCs may be seen resulting in leucoerythroblastic reaction
Howell Jolly bodies and Cabot’s rings may be seen
Leucopenia due to absolute neutropenia (but usually not less than 1,500/cmm)
Neutrophils are larger and have hypersegmented nuclei
When more than 5% of neutrophils have more than 5 lobes or at least one 6 lobed WBC in a film is present, it is called hypersegmentation.
Polymacrocyte is a large polymorph with nucleus containing up to 9 lobes
Hypersegmentation may also be found in case of renal failure, congenital form and in iron deficiency states.
Platelet count is moderately reduced (but not less than 40 x 109 /Lit)
Large forms of platelets are seen especially when counts are markedly reduced.
Bone marrow aspiration
Generally, not required if biochemical parameters are consistent with megaloblastic anemia.
Cellularity is increased (Rarely hypocellular)
Myeloid to erythroid ratio is 1:1
Erythroid precursors show megaloblastic changes
Cells are much larger than erythroblasts
Nucleus has fine, reticular chromatin which gives a stripped appearance (Loose open/ salt pepper granular chromatin) and large nucleoli.
Nucleocytoplasmic asynchrony- Maturation of nucleus lags behind that of cytoplasm (Nucleus is young and cytoplasm is mature).
Fully hemoglobinized orthochromatic erythroblasts which retain nuclei may be seen.
Howell-Jolly bodies (Fragmented nuclei) may be seen.
Abnormal and more common mitosis.
Maturation arrest is visible.
These changes disappear after 24hours of administration of vitamin B12 and folic acid supplementation
Megaloblastic marrow may also be seen in case of iron deficiency anemia, sideroblastic anemia, erythroleukemia, chronic anemia thalassemia, chromic infection etc
Masked megaloblastosis: Classic findings of megaloblastic changes may not be seen due to co-existing conditions the neutralize the tendency to generate megaloblastic cells. Ex: Iron deficiency anemia, thalassemia, anemia of chronic disease. There is a wide RDW in these conditions.
Increased iron granules on Prussian blue staining.
Leucopoiesis
Giant metamyelocytes (Giant stab) and bands with large horseshoe shaped nuclei containing loose, open chromatin in nuclei are diagnostic
Myelocytes show poor granulation
Large atypical granulocytes may be seen.
Megakaryocytes are abnormally large with multilobulated (hyperdiploid) nucleus and deeply basophilic agranular cytoplasm.
Microscopy of other cells: Epithelial cells of stomach, mouth and cervix may look megaloblastic. These cells appear larger than their normal counterparts and contain atypical immature looking nuclei. Distinguishing these "megaloblastic changes" from changes of malignancy can be difficult.
Reticulocyte count- Normal, but RPI is less than 2
Serum LDH level- Levels of fraction 1and 2 are markedly elevated. This is due to destruction of megaloblasts which are rich in LDH
Serum levels of vitamin B-12-
Decreased to less than 200pg/ml(Normal-200-350pg/ml)
It can be normal in clinically proven megaloblastic anemia
Holotranscobalamin assay (In this Vitamin B-12 bound to transcobalamine is measured). It represents 20-30% of total vitamin B-12
Serum folate assay:
Normal -3-25ng/ml
Rapid transport of sample is important, as folate is unstable and is destroyed by heat and light. (Adding sodium ascorbate stabilizes folate. But this sample cannot be used for assessment of vitamin B12.)
Red cell folate assay
Red cells contain 20-25 times more folate than in serum
This assay is more accurate, as serum levels fluctuate with dietary intake.
It is not useful in cobalamin deficiency, as in this case RBC folate is reduced.
Normal -340-640 ng/L of packed cells
Intrinsic factor antibody test: Positive in pernicious anemia
Schilling test
Radioactive vitamin B-12 is given orally followed 2hours later by high dose of parenteral B-12 injection.
Normally more than 10% of administered radioactive Vitamin B-12 is excreted in urine as large amount of parenteral unlabelled B-12 binds to most tissue binding sites.
Patients with malabsorption of Vitamin B-12 excrete <5% of administered radioactive-B-12
In such patients radioactive B-12 is again given orally along with intrinsic factor
If urinary excretion of radioactive Vitamin-B12 now rises to >10% it indicates patient is suffering from pernicious anemia.
Presently this test are not done due to
Reduced availability of test components
High cost
Problems with radioactive waste disposal
Concerns about use of animal derived tissues for human use (Intrinsic factor)
FIGLU excretion test
Folate is required for conversion of FIGLU (which is formed from histidine) to glutamic acid.
Large dose of histidine is given to the patient.
Increased excretion of FIGLU indicates folate deficiency
Serum gastrin level- Increased in pernicious anemia
Serum ferritin and iron levels- Increased due to large degree of ineffective erythropoiesis
Haptoglobins, Uric acid and alkaline phosphatase levels – Decreased
Serum methylmalonate and homocystein levels
Vitamin B-12 is needed for conversion of methylmalonate to SuccinylCoA and homocystein to methionine
So in Vitamin -B-12 deficiency methylmalonate and homocysteine levels are raised.
Other conditions in which homocysteine level is raised.
Chronic renal disease.
Small bowel bacterial overgrowth
Haemoconcentration
Alcoholism.
Smoking.
Pyridoxine deficiency.
Hypothyroidism.
Drugs – steroids, cyclosporine etc
Gastric biopsy- For pernicious anemia
Serum antiparietal cell antibody assay
Small intestinal studies- Duodenal biopsy to rule out celiac disease
Stool for fish tapeworm ova.
Pretreatment Work-up:
History
Examination
Hemoglobin
MCV
TLC, DLC
Platelet count
Peripheral smear
Reticulocyte count
S. B12 Levels
S. Folic acid level
Anti- IF antibody
LFTL Bili- T/D SGPT: SGOT:Albumin: Globulin:
Creatinine
LDH
Treatment:
Treat severely ill patient with both vitamins in large doses, once the blood sample has been taken for vitamin B-12 and folate assay.
Try to identify the cause by history, examination and investigations. If any cause is found treat it promptly.
Transfusion is not necessary, but if needed packed red cells should be given. If decompensated transfuse under cover of furosemide.
Platelet concentration should be given if there is spontaneous bleeding
Vitamin B-12 (Hydroxo/ cyanocobalamin)-
1000 micrograms – IM/IV
Daily for 5-7 days
Then- weekly for 5 weeks
Then- once in 3 months (If neurological deficits at presentation- once in 2 months)- Lifelong
Transient hypokalemia is often seen- Treat with oral potassium supplements
Reticulocytosis peaks at 1 week and becomes normal by 20th day.
Hemoglobin returns to normal in 5-6 weeks.
LDH falls to 50% in 72 hrs.
Normal pronormoblasts appear within 4-6 hours, and complete recovery of erythroid abnormalities occur within 2-4 days.
Hypersegmented neutrophils disappear after 15 days.
Peripheral neuropathy may be reversed, but spinal cord damage is irreversible.
In case of vitamin B-12 deficiency if folate alone is given it aggravates neurological damage. Hence avoid multivitamin preparations
Oral supplements are available and are useful even in cases of pernicious anemia. Dose- 1,000-2,000 micrograms/day
Folic acid
5mg/day-orally
Continue the therapy for 4 months, till all folate deficient RBCs are eliminated.
Treatment of causes:
Diphyllobothriumlatum: T. Praziquantel 5-10mg/kg- Single dose
Pyrimethamine, trimethoprime and most of other drugs- Folinic acid
Infants on specialized diets- On vegetarian diet- PO. 5-10microgam/day, if malabsorption- PO- 1000microgm/day (Absorption happens by passive diffusion)
Premature infants
Infants of mothers with pernicious anemia
Infants and children of mothers with nutritional cobalamin deficiency
Vegetarians and poverty induced near vegetarianism
Total gastrectomy
Folic acid:(Dose- 400micrograms/day)
All women contemplating pregnancy
Pregnancy and lactation
Premature infants
Mothers at risk of delivering infants with neural tube defects (Previous history of delivery of child with NTD. Dose- 5mg/day)
Patients with rhematoid arthritis/ psoriasis on therapy with methotrexate(Dose- 5mg/day)
Hemolytic anemia/ hyperproliferativehematological states (Dose- 5mg/day)
Neural tube folding occurs by 28 days, before even woman knows that she is pregnant. Hence better to give folate supplements to all those who are planning pregnancy.
Prophylaxis during pregnancy also prevents acute lymphoblastic leukemia in children
Related Disorders:
Pernicious anemia
It is commonly seen after 40 years of age.
It is characterized by autoimmune destruction of gastric mucosa leading to chronic atrophic gastritis.
3- Types of auto antibodies are found (IgG type).
Type
Character
Prevalence
1.
Blocking Ab
Blocks vitamin B-12 and IF binding
75%
2.
Binding Ab
Reacts with IF and prevents its attachment to ileal mucosa
50%
3.
Parietal CanalicularAb
Directed against α and β subunits of gastric proton.
90%
Risk Factors
?Genetic- More common in people with blood group A.
Other autoimmune disease – Grave’s disease, Hashimoto thyroiditis etc
Association with HLA-B8, B12, BW15.
?H. Pylori triggered autoimmune reaction
These patients are 3 times more prone to develop carcinoma of stomach
Investigations
Gastric biopsy
Loss of parietal cells and chief cells.
Prominent infiltration of lymphocytes and plasma cells.
Changes are marked in body of stomach
Metaplasia of gastric epithelium to intestinal type
Serum gastrin levels- Elevated
Serum Somatostatin levels- Decreased
Serumpepsinogen I levels-Decreased
Gastric function test-Histamine unresponsive achlorhydria
Serum antiparietal cells antibodies
They may be present also in cases of gastritis, thyroid disease and Addison’s disease
Treatment
Parenteral vitamin B-12 therapy
Follow up for early detection of gastric carcinoma
Steroids can be used but it is associated with increased risk of malignancy and improvement in gastric lesion is only temporary.
Juvenile pernicious anemia
It is a familiar condition, characterized by congenital deficiency of intrinsic factor.
It is commonly associated with endocrinopathies.
Infantile onset cerebral folate deficiency:
Develops 4-6 months after birth
Presents with agitation, insomnia, delayed development of head, psychomotor retardation, cerebellar ataxia, pyramidal tract signs in legs, dyskinesias, severe polyneuropathy, sometimes seizures
Pathogenesis: Infants fed on cow's milk develop autoantibodies to milk folate binding proteins. These antibodies bind to folate receptors in choroid plexus and block thefolate receptor mediated folate transport across the cerebrospinal fluid. This leads to cerebral folate deficiency
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