Hematology I (SCIE2020) PDF

Hematology I (SCIE2020) Harmening - Chapter 7 (5th Ed) Harmening – Chapter 8 (6th Ed) Megaloblastic Anemias 2 OBJECTIVES 4.1 Describe clinical signs of anemia 4.2 State the laboratory criteria for the diagnosis of anemia 4.3 State the significance of red blood cell indic...

Hematology I (SCIE2020) Harmening - Chapter 7 (5th Ed) Harmening – Chapter 8 (6th Ed) Megaloblastic Anemias 2 OBJECTIVES 4.1 Describe clinical signs of anemia 4.2 State the laboratory criteria for the diagnosis of anemia 4.3 State the significance of red blood cell indices as related to the diagnosis of anemia 4.9 Define anisocytosis and poikilocytosis and list clinical conditions in which they may be reported 4.10 Define the terms normochromic, hypochromic, microcytic, and macrocytic as they relate to red cell indices 4.11 Correlate red cell indices with red cell morphology and the diagnosis of anemia 4.20 Identify and describe the morphological alterations of size, shape, colour, and abnormal distribution patterns in erythrocytes 4.21 List any inclusions that may be found in erythrocytes 4.22 Compare the categories of anemia based on morphology 4.23 Describe the clinical presentation and laboratory findings of the following conditions: 4.23.2 Megaloblasitic anemia SUMMARY Anemia: Low Hgb or Hct level in the patient Macrocytic Anemias: Increased MCV > 100fL; “Macro” = “Large” RBCs with a big volume / size Megaloblastic Anemia One of the most common anemias worldwide It is a macrocytic anemia(MCV > 100 fL) The primary cause is defective DNA The DNA is not normal; The RBC grows beyond normal parameters – This includes large RBC cells in the bone marrow There are decreased total numbers of RBCs due to inter- medullary hemolysis; To compensate there’s erythroid hyperplasia in the bone marrow (aka. Fewer RBCs, but larger RBCs, to try and achieve a “normal” The mean corpuscular volume (MCV) is usually functionality) greater than 100 femtoliters (fL) with macrocytic anemia. Megaloblastic Anemia  Megaloblastic anemia is a subgroup of macrocytic anemia (MCV >100 fL) characterized by defective nuclear maturation caused by impaired deoxyribonucleic acid (DNA) synthesis  This is often caused by dietary deficiencies (e.g. Vitamin B12 and/or Folate)  This defect is manifested by the presence of  Megaloblasts (large and abnormal red cell precursors) in the bone marrow  Macro-ovalocytes in the peripheral blood  Hyper-segmented neutrophils (aka. Polymorphonuclear or PMNs) with > 5 lobes, which means there’s more than 5 pieces of the nucleus – “megaloblastic” In B12 and folate deficiency, there’s impaired or disrupted synthesis of DNA there’s fewer cell divisions; the nucleus lags behind the cytoplasm aka. nuclear-to- cytoplasm asynchrony Chromosomes may become separated in cell division, forming Howell- Jolly Bodies 7 Megaloblastic Anemia So, because the mechanism is impaired production of DNA, the other cell lines (in additional to the RBCs) are affected too.  Hyper-segmented neutrophils– greater than 5 lobes (aka. Polymorphonuclear, or PMN)  Also, granulocyte precursors tend to be larger than normal, with giant metamyelocytes  In severe anemia, an abnormal nuclear pattern in megakaryocytes may be seen Biochemical Aspects  The defective nuclear maturation and the megaloblastic morphology are caused by a decrease in thymidine triphosphate (TTP) synthesis  (Deoxythymidine triphosphate is one of the four triphosphates that are used in the in vivo synthesis of DNA). This deficiency interferes with nuclear maturation, DNA replication, and cell division.  When TTP is not present in adequate amounts – there’s fragmentation of the nucleus and ultimately destruction of immature cells.  Vitamin B12 and folic acid deficiencies - these vitamins, in the form of cofactors, play important roles in some key reactions involved in DNA synthesis.  Also, drugs that interfere with the metabolism of these vitamins may also cause DNA impairment. Clinical Manifestations of Megaloblastic Anemia  Certain clinical manifestations are common to all patients with megaloblastic anemias regardless of the cause.  The degree of anemia may be mild to severe, with the symptoms of weakness, fatigue, shortness of breath, and lightheadedness. Congestive heart failure may or may not be present, depending on the degree of anemia.  In severe anemia, the patient may have a lemon-yellow skin tint because of mild jaundice and pallor. Increased bilirubin is reported in about 30% of patients as a result of intramedullary hemolysis caused by ineffective erythropoiesis. Hematologic Features Ineffective Hematopoiesis  Megaloblastic anemia is associated with ineffective erythropoiesis and hemolysis.  Generally, patients with megaloblastic anemia may have MCV values > 130fL (and possibly as high as 160 fL). This elevated MCV reflects the megaloblastic picture of the bone marrow.  Decreased red cell release into the peripheral blood is indicative of ineffective erythropoiesis which is supported by decreased reticulocytes (i.e. reticulocytopenia). Ineffective Hematopoiesis  CONTINUED Megaloblastic erythrocyte progenitors have a much shorter life span than normal erythrocyte progenitors.  They are more fragile and, therefore, die prematurely in the marrow.  Evidence of intramedullary hemolysis includes decreased haptoglobin, increased levels of serum bilirubin, serum lactate dehydrogenase (LD, in particular, LD-1 and LD-2 isomers), and increased serum iron.  Cell death occurs primarily at the later stages of the megaloblast maturation (i.e., basophilic and polychromatophilic stages), causing a decrease in production /release of mature erythrocytes.  A decreased level of erythrocytes in the circulation stimulates erythropoietin release, which in turn stimulates production of red cell progenitors. Ineffective Hematopoiesis  CONTINUED  Ineffective granulopoiesis is defined by increased bone marrow white cell precursors and failure to release mature forms into the peripheral blood. The giant cells do not mature to circulating neutrophils but, rather, die prematurely in the bone marrow.  Bone Marrow: Megaloblastic changes are manifested in white cell precursors by the presence of large/giant bands (see fig 7- 2B and next slide) and giant metamyelocytes in the bone marrow.  Ineffective thrombopoiesis possibly - may show the presence of increased abnormal megakaryocytes in the bone marrow and thrombocytopenia in the peripheral blood. Giant band Bone Marrow Morphology  Patients with megaloblastic anemia have a hypercellular bone marrow.  The myeloid-to-erythroid (M:E) ratio is decreased (see fig 7-2 next slide) because of an increase in erythroid precursors.  The degree of increased cellularity (megaloblastic picture) depends on the severity of the anemia.  Bone Marrow Morphology Megaloblasts are large cells with increased RNA.  Their nuclear chromatin appears loose and less mature than the nuclear chromatin of the normal red cells at the same stage of maturation (recall slide #7 and see fig 7-3 next slide). The cytoplasm maturation is, however, typically normal. This phenomenon is referred to as nuclear-to-cytoplasm asynchrony.  The mature megaloblastic red cells entering the circulation usually have a shorter life Source: Harmening Figure 7-2 A. Mitotic figures in span than normal mature red cells. megaloblastic marrow. B. Large megaloblastic band neutrophil. C. Megaloblastic pronormoblast with open, sievelike chromatin. The myeloid-to- erythroid (M:E) ratio is decreased because of the increase in megaloblastic erythroid precursors labeled A and C., p. 140  H ow el lJ ol ly Bo di se Source: Harmening Figure 7-3 Bone marrow. A. Polychromatophilic megaloblasts. B. Orthochromic megaloblast with multiple Howell–Jolly bodies, p. 140 Peripheral Blood Morphology  Megaloblastic anemia is a macrocytic, normochromic anemia.  Increased MCV: depending on the degree of anemia; the MCV is elevated (may range from 100fL to 160 fL). The mean corpuscular hemoglobin concentration (MCHC) is usually normal. NOTE: Not ALL patients with macrocytosis have megaloblastic anemia (!) For example, alcoholism and liver disease NOTE: Also, not ALL megaloblastic anemias are macrocytic! For example, a normal MCV may possibly be present in patients with megaloblastic anemia and coexisting iron deficiency (dimorphic). Peripheral Blood Morphology  The hemoglobin or hematocrit value is expected to be low. In a severe anemia the hemoglobin may drop to quite low, for example 70 to 80 g/L.  The erythrocyte count is generally decreased.  In patients with untreated megaloblastic anemia, the macrocytes have a shortened survival time (27 to 75 days) compared with the survival time of normal red cells.  The leukocyte count may be normal at the early stage of anemia but may decline eventually.  Although platelets are the least affected cell line, platelet counts below 100 × 109/L have been reported in patients with severe anemia. Peripheral Blood Morphology  The peripheral blood smear has macrocytes and macro- ovalocytes and possibly pancytopenia  The degree of anisocytosis and poikilocytosis varies with the severity of anemia.  Other poikilocytes, such as schistocytes, teardrop-shaped cells, spherocytes, and target cells, may also be seen on the peripheral blood smear.  Increased anisocytosis causes an elevated red cell distribution width (RDW) as determined via an automated cell counter.  As mentioned, dimorphic red cell morphology may be present in patients who have a co-existing condition, such as iron-deficiency anemia, thalassemia, anemia of chronic disease, or hyperthyroidism in addition to the megaloblastic anemia. Peripheral Blood Morphology  Red cell inclusions such as Howell–Jolly bodies are frequently present (see fig 7-5 next slide) and possibly basophilic stippling. Cabot rings and megaloblastic nucleated red cells may be seen on the peripheral blood smear (see slide ahead fig 7-6).  The absolute reticulocyte count is decreased with a reticulocyte production index (RPI) of less than 2, indicating ineffective erythropoiesis. With treatment, the number of reticulocytes increases along with increased numbers of nucleated red cells. Source: Harmening Figure 7-5 Howell–Jolly body in an RBC in pernicious anemia (arrow), p. 141 Source: Harmening Figure 7-6 Cabot ring in pernicious anemia (arrow), p. 141 Source: Harmening Figure 7-4 Extreme degree of anisocytosis (+4) and poikilocytosis (+4) with oval macrocytosis (arrow) in a patient with severe pernicious anemia, p. 140   Multi-lobed neutrophils, termed “hypersegmented neutrophils”, are often seen in the peripheral smear (also see fig 7-7 next slide)  Hyper-segmented neutrophils refer to neutrophils > 5 lobes NOTE: One hyper-segmented cell is NOT SIGNIFICANT! MLTs should see 5-OR-MORE GRANULOCYTES WITH 5-OR-MORE LOBES before reporting “hyper- segmented” cells seen. Source: Harmening Figure 7-7 Neutrophil hypersegmentation in pernicious anemia, p. 141  The diagnosis of megaloblastic anemia may be based on the morphological characteristics of the peripheral blood and the results of other biochemical lab tests.  Bone marrow examination is generally not required The clinical features of megaloblastic anemia are summarized in table 7-1 (next slide). Laboratory Tests: See Table 7-1 next slide  Elevated lactate dehydrogenase (LD) – due to hemolysis  Indirect bilirubin and urobilinogen  Decreased haptoglobin  Increased serum iron and serum ferritin  Increased erythropoietin (EPO) Source: Harmening Table 7-1 p. 141 STRUCTURE and ABSORPBTION Vitamin B12 (cobalamin) is a large, water-soluble molecule. It is absorbed in the terminal ileum. B12 has a special way of being absorbed – parietal cells in the stomach release intrinsic factor (IF) and IF binds with B12 and travels along with it all the way down the gut, to the terminal ileum. Only when they’re attached together, will B12 get absorbed. So, IF is needed in order to absorb vitamin B12! Vitamin B12 Deficiency SOURCES AND REQUIREMENTS  Vitamin B12 is produced by microorganisms and fungi.  It is present in foods of animal origin such as liver, fish, poultry, meat, eggs, and dairy products. Liver is a major source of vitamin B12. Vegetables do not contribute B12 to the diet.  Vitamin B12 is commercially available as a supplement for treatment of deficiencies.  Vitamin B12 Deficiency REQUIREMENTS  The recommended dietary intake of vitamin B12 for adults is 5 µg/day.  This requirement increases in pregnancy, infancy, during growth, for example.  Vitamin B12 is lost through the urine and feces. Body storage of vitamin B12 is about 1 to 5 mg, of which approximately 1 to 2 mg know is stored in the liver (some references say up to 3mg). Approximately 3-10 years of storage in the liver.  Because the daily requirement of vitamin B12 is low and the storage rate is high, it takes several years for a person to develop vitamin B12 deficiency as a result of malabsorption (i.e. It takes years to develop a deficiency! This is due to large hepatic stores and small daily loss).  Harmening p. 142 Two important proteins are involved in the transport of vitamin B12 from the duodenum to the ileum and from ileum to tissues: the intrinsic factor (IF) and transcobalamin II. Gastric parietal cells in the stomach produce IF IF is a glycoprotein B12 combines with IF produced by the parietal cells of the stomach; it combines with vitamin B12 on the Ileum is the final portion of the small intestine ileum of the small transcobalami intestine; so, IF n helps the intestines absorb vitamin B12 TC II is a protein that carries vitamin B12 in plasma Source: Harmening Figure 7-9 Transportation path of vitamin B 12 from the diet to the tissues. IF = 33 intrinsic factor; TC II = transcobalamin II, p. 142 TRANSPORT AND METABOLISM  Two important proteins are involved in the transport of vitamin B12 from the duodenum to the ileum and from ileum to tissues: the intrinsic factor (IF) and transcobalamin II.  Dietary cobalamin is released from the food by gastric acids and intestinal enzymes. On release, it binds to a carrier protein called R protein.  On entering the duodenum, B12 releases from the R protein by the action of pancreatic enzymes. The released vitamin B12 then binds to IF, a glycoprotein.  The gene for IF is located on chromosome 11.  IF is secreted by parietal cells of the stomach. The parietal cells also secrete hydrochloric acid and other gastric juices. IF binds to vitamin B12 and forms B12–IF complex, which allows vitamin B12 to be absorbed through the receptors present on the ileum (the distal half of the small intestine) see fig 7-9.  IF is not absorbed by the ileum and, therefore, cannot be reutilized. It is degraded on release from vitamin B12. The released vitamin B12 then enters the portal vein. TRANSPORT AND METABOLISM  When vitamin B12 leaves the ileum and enters the portal vein, it attaches to three proteins—transcobalamin I (TC I), transcobalamin II (TC II), and transcobalamin III (TC III).  About 70% to 90% of vitamin B12 is bound to TC I, TC III, and other R proteins, whereas only about 10% to 25% of vitamin B12 is bound to TC II.  TC II is the main transport protein of cobalamin to the tissues. It is a polypeptide.  TC II is synthesized by ileal cells, endothelial cells, liver, spleen, heart, and macrophages, and is secreted into the plasma.  TC II transports vitamin B12 to the liver for storage and to the bone marrow and other tissues for DNA synthesis (see fig 7-9).  Deficiency of TC II causes megaloblastic anemia.  It is suggested that the functions of TC I and TC III are to bind to vitamin B12, preventing its losses in the urine. DIETARY VITAMIN B12 DEFICIENCY  Nutritional vitamin B12 deficiency and strict vegetarians: In this group, the decrease in vitamin B12 is accompanied by an increased plasma folate level.  The major cause of vitamin B12 deficiency is malabsorption. The most common form of intestinal malabsorption is pernicious anemia. Other causes of vitamin B12 deficiency are summarized in Table 7-2 (next slide). Source: Harmening Table 7-2, p. 143  Megaloblastic Anemia *  B12 deficiency  Folic Acid deficiency  Anti-folate drugs  Cancer/chemotherapy * Partial list ONLY, relevant for MLTs PERNICIOUS ANEMIA PATHOPHYSIOLOGY  A main cause of pernicious anemia is atrophic gastritis characterized by atrophy of the gastric mucosa with decrease of gastric secretions and gastric intrinsic factor. The cause of gastric atrophy is probably autoimmune, with poorly defined genetic predisposition.  Gastric intrinsic factor is essential for absorption of vitamin B12. In the absence of gastric intrinsic factor, only a small amount of vitamin B12 is absorbed, causing a gradual deficiency in vitamin B12.  (This is for information purposes – You don’t need to know all the details here ) Genetic Factors  The exact cause of the genetic predisposition of pernicious anemia is not yet clear. A weak association has been made between pernicious anemia and the human leukocyte antigen (HLA), but it is not conclusive.  An association between pernicious anemia and other autoimmune diseases, such as thyroid disease, diabetes mellitus, and rheumatoid arthritis, has also been noted. The positive response to steroids in some patients with pernicious anemia supports the autoimmune mechanism.  The Helicobacter pylori microorganism has been identified as a major cause of gastritis and peptic ulcers. Immunologic Factors  The serum of patients with pernicious anemia contains autoantibodies to parietal cells, to gastric intrinsic factor, and to thyroid tissue. This antibody seems to be specific for the parietal cells only. CLINICAL MANIFESTATIONS OF VITAMIN B12 DEFICIENCY  The onset of pernicious anemia is generally gradual/insidious.  Patients with pernicious anemia and other vitamin B12 deficiencies have all the signs and symptoms of megaloblastic anemia mentioned earlier.  Fever may be present in severe anemia.  Loss of appetite is a common complaint.  Glossitis (sore tongue) is reported in approximately 50% of patients. Although the initial presentations may vary among patients with vitamin B12 deficiency, the classic symptoms include weakness, glossitis, and paresthesias (tingling/numbing in hands, arms, legs or feet). CLINICAL MANIFESTATIONS OF VITAMIN B12 DEFICIENCY The bone marrow morphology of patients with vitamin B12 deficiency is megaloblastic and the peripheral smear contains macro-ovalocytes.  In addition to hematologic abnormalities, vitamin B12 deficiency is associated with gastrointestinal, thrombotic and neurologic complications. NEUROLOGIC MANIFESTATIONS  Neurologic problems are more common in pernicious anemia than in other types of vitamin B12 deficiencies.  The neurologic abnormalities may be mild, moderate, or severe and may involve degeneration of peripheral nerves and the spinal cord (see table 7-3). Source: Harmening p. 145 NEUROLOGIC MANIFESTATIONS  In the earlier stage of pernicious anemia, the peripheral nerves are affected.  The patient often experiences symmetric tingling or “pins-and-needles” sensations in the toes and later in all four limbs.  At the later stage, the posterior spinal columns may be involved. At this stage, the patient may complain of clumsiness and have an incoordinate gait. The most severe stage of illness, with manifestations of severe weakness and stiffness of limbs, have impairment of memory and depression.  Severe psychiatric symptoms are less common and include hallucinations, and severe depression.  Neurologic manifestations of less than 3 months' duration are usually reversible.  In untreated patients, the neurologic symptoms are progressive, and the degree of severity is directly proportional to the duration of symptoms. OTHER CAUSES OF VITAMIN B12 DEFICIENCY (This is for information purposes – You don’t need to know all the details here ) GASTRECTOMY -Many other causes of malabsorption can lead to vitamin B12 deficiency (see Table 7-2). In a gastrectomy procedure, the IF-producing cells are removed. In the absence of vitamin B12 therapy, vitamin B12 deficiency develops in these patients within several years. Vitamin B12 deficiency has been reported in 30% to 40% of patients with partial gastrectomy. BLIND LOOP SYNDROME -In blind loop syndrome, an anatomic abnormality of the small intestine, there is an overgrowth of bacteria in the small bowel. These microorganisms take up the vitamin B12 and make it unavailable for absorption by the ileum. Tetracycline therapy for 10 days normalizes the vitamin B 12 level. FISH TAPEWORM -Fish tapeworm (Diphyllobothrium latum) is a parasite that competes for vitamin B12 by splitting B12 from The malabsorption type of vitamin B12 deficiency is normally corrected when vitamin B12 or B12 and IF are given to the patients. DISEASES OF ILEUM -Vitamin B12 deficiency can also be seen in diseases of the ileum, for example, after ileal resection. OTHER CAUSES OF VITAMIN B12 DEFICIENCY (This is for information purposes – You don’t need to know all the details here ) CHRONIC PANCREATIC DISEASE - In pancreatic disease, vitamin B12 deficiency develops as a result of a decrease in the proteases necessary for release of vitamin B12 from salivary and gastric R proteins for absorption. A low level of free calcium, which is necessary for calcium-dependent ileal absorption, can cause vitamin B12 deficiency in patients with chronic pancreatic disease. OTHER DISORDERS - Vitamin B12 deficiency has also been reported in patients who are on hemodialysis and in patients with human immunodeficiency virus (HIV) infection and with acquired immunodeficiency syndrome (AIDS). DRUG-INDUCED VITAMIN DEFICIENCY - Other causes of vitamin B12 deficiency are drugs such as alcohol, anesthetics, nitrous oxide (N2O), and the antituberculosis drug para- Folic Acid Deficiency SOURCES AND REQUIREMENTS Folic acid is a water-soluble vitamin present in a variety of foods. Highest concentration is present in green leafy vegetables, fruits, dairy products, cereals, and also in animal foods, such as liver and kidney. The average daily diet contains about 400 to 600 µg of folate; however, folate is a heat-labile vitamin and, therefore, is easily destroyed in overcooked vegetables. The recommended dietary intake of folic acid for adults is approximately 50 to 100 µg/day. Is absorbed in the duodenum.  Folic Acid Deficiency SOURCES AND REQUIREMENTS This requirement increases significantly during infancy, pregnancy, and lactation. Folate deficiency during early pregnancy (first trimester) can cause renal tube defects (NTDs) in the fetus and is associated with paralysis and brain damage. The body storage is about 5 to 10 mg, of which most is stored in the liver. Folic acid is absorbed through the duodenum and jejunum, and the amount absorbed is about 80% of intake. Folate is lost via body secretions such as bile, urine, and sweat. Folic acid has a higher turnover time and a higher rate of loss compared to Vitamin B12 and, therefore, it takes only a few weeks/months to know develop dietary folate deficiency. So, folate deficiency usually develops within weeks/months because the body stores are minimal.  DIETARY DEFICIENCY  The main cause of folic acid deficiency is decreased dietary intake/ poor diet.  Other causes are malabsorption (GI disease such as Crohn’s or celiac; cancer), CAUSES increased requirement (pregnancy/lactation), and drug-induced folate deficiencies (for example OF FOLIC methotrexate, a chemotherapy drug which blocks DNA synthesis) See table 7- 4. ACID  Folic acid has been added to cereal grains, rice, and milled flour to increase one’s intake DEFICIENC  Women planning to become pregnant and in early pregnancy may be advised to eat Y a diet rich in folic acid, or take vitamin supplements, to prevent the adverse effects of folic acid deficiency in the developing fetus.  Folate deficiency can cause serious birth defects in the spinal cord and brain of a developing fetus, which are called neural tube defects (NTDs). CLINICAL MANIFESTATIONS OF FOLIC ACID DEFICIENCY  Clinical manifestations of folate deficiency are the same as those for Vitamin B12 deficiency, mentioned earlier.  The onset of anemia is insidious, with the distinct morphology characteristic of megaloblastic anemia in the bone marrow and in the peripheral blood.  Although neuropathy is mainly characteristic of vitamin B12 deficiency, several cases of neurologic abnormalities, such as depression, dementia, and peripheral neuropathy associated with folic acid deficiency, have been reported. Some of these neuropathies, in particular depression, have responded favorably to treatment with folate. Laboratory Diagnosis of Megaloblastic Anemia  Several important factors in differential diagnosis of megaloblastic anemias are the patient's physical examination, medical history, drug history, family history, and laboratory tests.  We can test the folate level – would be LOW  The most common laboratory screening tests and results that are used in the diagnosis of megaloblastic anemias are:  Low hemoglobin level or Hematocrit  High (elevated) MCV  Peripheral smear morphology, such as macro-ovalocytes and hypersegmented neutrophils. Once the diagnosis of megaloblastic anemia is established, the exact cause of the anemia should be determined for appropriate and effective treatment. Source: Harmening Table 7-5, p. 148 SCHILLING TEST  The Schilling test evaluates the pathophysiology of vitamin B12 malabsorption. The test is done in two parts.  In Part 1, the patient is given 0.5 to 2.0 µg of labeled (57Co or 58 Co) vitamin B12 orally.  Two hours later, a flushing dose (1000 µg) of unlabeled vitamin B 12 is injected intramuscularly to saturate all of the circulating cobalamin binders.  The amount of the labeled vitamin B12 is then measured in a 24-hour urine collection.  If IF is present and normal absorption takes place, the labeled vitamin B12 absorbed through the intestine is rapidly excreted into the urine.  The urinary excretion varies, depending on the dosage given.  In normal absorption, about 5% to 35% of labeled B12 is excreted in the urine. An abnormal result in Part 1 indicates that B12 was not absorbed through the intestine.  In this case, testing proceeds to Part 2 to find the cause of malabsorption (see Fig. 7–15). SCHILLING TEST  In Part 2, the test is repeated with the addition of IF to the oral dose to determine if malabsorption is caused by the lack of IF.  If the Schilling test is corrected in Part 2, a deficiency of IF is confirmed. If the Schilling test is still abnormal, other causes of malabsorption should be investigated (see Fig. 7–15).  Reliability of the Schilling test depends on normal renal function and proper urine collection. The Schilling test is difficult to perform; therefore, many hospital laboratories no longer offer the test.  Some investigators think that the measurement of serum cobalamin binding protein (holoTC II) may become a good replacement for the Schilling test. SCHILLING TEST The Schilling test evaluates the pathophysiology of vitamin B12 malabsorption. The test is done in two parts Source: Harmening Figure 7-15 The two-part Schilling test. IF = intrinsic factor, p. 149 Treatment of Megaloblastic Anemia Vitamin B12 Deficiency  Most people with a vitamin B12 deficiency require lifelong vitamin therapy. Cyanocobalamin and hydroxocobalamin are the two therapeutic forms of vitamin B12.  Vitamin B12 can be administered orally to patients with dietary vitamin deficiency or to those who cannot tolerate parenteral treatment.  Vitamin B12 is injected intramuscularly or subcutaneously. The treatment protocol varies, Vitamin B12 may be given as 100 to 1000 µg/day for 2 weeks, then weekly until hematologic values are normalized and then monthly for life. Vitamin B12 therapy may be monitored by reticulocyte counts. Folic Acid Deficiency  The recommended therapeutic dose to treat folate deficiency is 1 to 5 mg/day for 2 to 3 weeks. Folic acid vitamin is water soluble and is given orally. Lifelong therapy is not required because it is usually possible to treat folate deficiency within a short period of time.  It is important to treat the underlying cause, for example Chron’s disease.  Folic acid given as prophylaxis: (0.25 to 0.5 mg/day) is recommended during pregnancy and dialysis and may be required in patients with hemolytic anemia and in patients who are on anti-folate drugs. Folic acid can be injected to hospitalized patients and in those who cannot take the medication by mouth. Response to Therapy  The initial sign of a positive response to therapy is an increase in the reticulocyte count. The number of circulatory reticulocytes increases 3 to 5 days after therapy, with a peak at about 4 to 10 days. The reticulocyte count may increase to 50% to 70% initially.  The megaloblastic morphology of the bone marrow disappears within 24 to 48 hours after therapy. The hematocrit rises in about 5 to 7 days after therapy, reaching normal levels in 4 to 8 weeks. Giant metamyelocytes and hypersegmented neutrophils disappear within 2 weeks. The entire therapeutic response process may take only 3 to 6 weeks, depending on the severity of the disease.  Macrocytic Non-Megaloblastic Anemias  Non-Megaloblastic Anemia*  Liver disease  Alcoholism  Reticulocytosis – reactive – such as blood loss; pregnancy  Hypothroidism  Myelodysplastic syndrome  Chronic obstructive pulmonary disease * Partial list, relevant to MLTs Source: Harmening Table 7-7, p. 152 Causes of Macrocytic Non-Megaloblastic Anemias  The most common causes of macrocytic anemia – non-megaloblastic -- are chronic liver disease and alcoholism.  With alcoholism, macrocytosis is present in the absence of anemia, and in this case, alcohol has a direct toxic effect on the red cells rather than causing folate deficiency.  The finding of macrocytosis is a valuable screening test for early detection of alcoholism. Liver function tests are helpful in the diagnosis. Keep in mind that macrocytic anemias may be megaloblastic or non- megaloblastic Differentiation between the two is important (!)  In macrocytic, normoblastic anemias:  The MCV is more than 100 fL, but not as high as in megaloblastic anemias.  E.g. MCV = 105 fL  Indicative of a macrocytic, normoblastic anemia  E.g. MCV = 120 fL  Indicative of macrocytic, megaloblastic anemia  The red cells on the peripheral blood smear appear large and round, but not usually oval.  The neutrophils are not hypersegmented.  The bone marrow is normocellular or hypercellular with erythroid hyperplasia. The red cell precursors in the marrow are normoblastic and not megaloblastic.  The mechanism responsible for the macrocytic morphology may be associated with an increase in both red cell membrane cholesterol and phospholipid. Increased lipid deposition onto the red cell membrane and altered maturation time of the red cell precursors are among the possible causes. SOME SUMMARY POINTS ▪ Megaloblastic anemia is a macrocytic anemia characterized by defective nuclear maturation caused by impairment of DNA synthesis. ▪ Megaloblastic anemia is associated with ineffective erythropoiesis, ineffective granulopoiesis, and ineffective thrombopoiesis. ▪ The bone marrow of patients with megaloblastic anemia is hypercellular with a low M:E ratio (1:1 to 1:3), high number of megaloblasts, and giant bands and metamyelocytes. ▪ The peripheral blood is characterized by pancytopenia, macrocytes, macro-ovalocytes, and hypersegmented neutrophils. ▪ Other biochemical changes are increased levels of LDH, indirect bilirubin, serum iron and ferritin, and erythropoietin. SOME SUMMARY POINTS  CONTINUED ▪ The major causes of megaloblastic anemias are vitamin B12 deficiency, folic acid deficiency, or both. ▪ Vitamin B12 and folic acid in the form of cofactors are essential for two key reactions in the body. ▪ Intrinsic factor enhances vitamin B12 absorption through the receptors present on the brush borders of the ileum. ▪ ▪ The main cause of vitamin B12 deficiency is pernicious anemia. ▪ Pernicious anemia is the lack of gastric intrinsic factor. ▪ Clinical manifestations that are often associated with vitamin B12 deficiency are anemia, fever, glossitis, and neurologic symptoms. ▪ Other causes of vitamin B12 deficiency are dietary malabsorption secondary to diseases and drugs. ▪ The main cause of folic acid deficiency is a poor diet. ▪ Other causes of folic acid deficiency are malabsorption, increased requirement, and drugs. SOME SUMMARY POINTS  CONTINUED ▪ Clinical manifestations associated with folic acid deficiency are similar to those in vitamin B12 deficiency, with neuropathies not being the prominent features. ▪ Laboratory tests used for the differential diagnosis of vitamin B12 and folate deficiencies are serum B12, and serum and red cell folate. ▪ Other laboratory tests that may be useful are gastric achlorhydria, antibodies to intrinsic factor, Schilling test, etc. Vitamin B12 deficiency can be treated with cyanocobalamin or hydroxocobalamin, and folate deficiency can be treated with folic acid supplementation. ▪ The initial response to therapy is increased reticulocyte counts. ▪ Vitamin-independent megaloblastic anemias can be inherited or acquired. ▪ Macrocytic nonmegaloblastic anemias are characterized by a high MCV, macrocytes in the peripheral blood, and normocellular or hypercellular bone marrow with erythroid hyperplasia. ▪ The most common causes of macrocytic anemia are liver disease and alcoholism. LAB THIS WEEK Peripheral Blood Smear Analysis – Part I My Additional Notes Harmening Chapter 7 My Additional Notes Harmening Chapter 7