Identificamos nuestros propios activos de salud

With the help of the information gained from the clinical data and these basic laboratory studies, further investigations can be undertaken to define the underlying cause of anaemia.

Evaluation of Macrocytic Anaemias In macrocytic anaemia MCV is greater than 100 fl. In most cases, various causes of macrocytic anaemia can be differen-tiated on the basis of reticulocyte count and examinations of peripheral blood smear and bone marrow (Fig. 2.5).

Two types of macrocytosis can be

distinguished on blood smear: round and oval. Their causes are listed in Box 2.6.

Typical features of megaloblastic anaemia due to deficiency of vitamin B₁₂ or folate are—(1) Peripheral blood smear: macrocytic anaemia, leucopenia, and thrombocytopenia (pancytopenia); marked anisopo ikilocytosis (variation in size and shape of red cells);

Howell-Jolly bodies; and hypersegmented neutrophils (5 or more lobes in more than 5% neutrophils); (2) Bone marrow examination: Bone marrow examination confirms the diagnosis of megaloblastic anaemia. It shows ineffective erythropoiesis (increase in early erythroid precursors due to premature destruction of more mature erythroid

Figure 2.4: A simplified approach for evaluation of anaemia based on complete blood count

Box 2.6 Oval and round macrocytosis Oval macrocytosis

Megaloblastic anaemia due to deficiency of folate or vitamin B₁₂, Drug therapy (hydroxyurea, zidovudine, chemotherapy), myelodysplasia

Round macrocytosis

Alcoholism, liver disease, hypothyroidism

cells resulting in anaemia), megaloblasts with nuclear cytoplasmic asynchrony (nuclear chromatin is open or sieve-like while cytoplasm shows haemoglobinisation), and presence of giant bands and metamyelocytes. The distinction between folate and vitamin B₁₂ deficiencies is based on estimation of serum and red cell folate and serum vitamin B₁₂. Therapeutic trial can also be given to distinguish between the two deficiencies (See chapter on megaloblastic anaemias).

Reticulocytosis in haemolytic anaemias is another cause of macrocytosis. As reticulocytes are larger than mature red cells MCV is increased. Chronic extravascular haemolysis is associated with mild icterus, variable splenomegaly, and unconjugated hyperbilirubinaemia. Peripheral smear shows polychromatic cells and normoblasts.

Intravascular destruction of red cells is associated with haemoglobinaemia, haemoglobinuria, and haemosiderinuria.

Macrocytosis in liver disease is uniform, round, and is associated with target cells and abnormal liver function tests.

Most patients with myelodysplastic syndrome are elderly and have bi- or pan cytopenia. Bone marrow examination reveals dysmyelopoiesis and sometimes abnormal localization of immature precursors.

In alcoholic patients, macrocytosis can occur in the absence of megaloblastic marrow or alcoholic cirrhosis. The mechanism is unknown.

Macrocytosis also occurs in pregnancy, newborns, during cytotoxic chemotherapy and in aplastic anaemia.

Evaluation of Microcytic Hypochromic Anaemia

Causes of microcytic hypochromic anaemia are listed in Table 2.5. The most common cause of microcytic hypochromic anaemia is iron deficiency. In early stages of iron

Figure 2.5: Evaluation of macrocytic anaemia

severity of anaemia. The biochemical parameters of iron deficiency are low serum iron, increased total iron binding capacity (TIBC), low transferrin saturation (<15%), and low serum ferritin (<12 µg/L). Bone marrow examination shows micronormoblastic erythropoiesis and on prussian blue staining absence of stainable iron.

In b thalassaemia major, severe anaemia develops during first few years of life that requires regular blood transfusion therapy. Hepatosplenomegaly is present.

Peripheral blood smear shows marked anisopoikilocytosis, severe microcytosis and hypochromia, frequent target cells, basophilic stippling, and normoblasts.

Haemoglobin electrophoresis shows predominance of HbF.

In b thalassaemia minor, anaemia is either absent or mild and peripheral blood smear shows prominent red cell abnormalities such as microcytosis, hypochromia, basophilic stippling, and target cells. Haemoglobin electrophoresis typically shows increase in HbA2 (3.5–7%).

Sideroblastic anaemia exhibits dimorphic population of red blood cells in peripheral blood (normocytic normochromic and microcytic hypochromic) and ringed sideroblasts in bone marrow.

Patient may be evaluated for anaemia of chronic disease if there is a history of chronic inflammation, chronic infection or malignant disease. The anaemia is usually mild to moderate, serum iron and total iron binding capacity are reduced, serum ferritin is elevated, bone marrow morphology is normal and storage iron in marrow is normal or increased. Erythrocyte sedimentation rate is raised and does not correspond with the degree of anaemia.

A scheme for evaluation of microcytic hypochromic anaemia is presented in Figure 2.6.

Figure 2.6: Evaluation of microcytic hypochromic anaemia

Evaluation of Normocytic Normochromic Anaemia

Depending upon bone marrow erythropoietic activity, normocytic anaemias are divided into two types (Fig. 2.7 and Table 2.5).

Normocytic anaemias with increased reticulocyte count—Two possible causes are acute blood loss and haemolysis.

1. Acute posthaemorrhagic anaemia: Acute blood loss can occur either externally or internally (e.g. haemothorax, fracture of hip). Significant blood loss occurring rapidly over a short period of time causes acute blood loss anaemia.

After haemorrhage, to compensate for hypovolaemia, there is an increase in plasma volume due to movement of fluid from extravascular sites. This causes haemodilution and fall in haematocrit and haemoglobin levels. Anaemia does not become evident for 1 to 3 days after haemorrhage due to the time needed for restoration of plasma volume. Acute posthaemorrhagic anaemia is normocytic and normochromic. Stimulation of bone marrow by erythropoietin causes erythroid hyperplasia. Reticulocytosis begins about 3 days after the episode and reaches its peak around 9 to 10 days. During this period, nucleated red cells may appear in peripheral blood. Thrombocytosis and neutrophilic leucocytosis with mild shift to left (i.e. increase in immature white blood cells) are common findings. If haemorrhage is internal, destruction of extravasated red cells and catabolism of haem cause increase in serum bilirubin. If internal haemorrhage is not detected, these findings may be misinterpreted as indicative of haemolytic anaemia.

2. Haemolytic anaemia: Once the possibility of blood loss is ruled out, haemolytic anaemia is the prime consideration. Haemolytic anaemias are due to increased

Figure 2.7: Evaluation of normocytic anaemia

compensate for the increased rate of red cell destruction.

Tests to establish the presence of haemolysis: Various laboratory tests are used to detect haemolysis.

Red cell destruction can occur either extra-or intravascularly (Figs 2.8 and 2.9, and Table 2.7). Extravascular destruction of red cells by macrophages occurs mostly in spleen and liver. This leads to unconjugated hyperbilirubinaemia. Increased level of serum unconjugated bilirubin also occurs in other conditions such as ineffective erythropoiesis, internal haemorrhage, and certain liver disorders. It is, therefore, not a specific marker of haemolysis.

Serum lactate dehydrogenase level rises due to the release of the enzyme from the haemolysed red cells. Raised levels of lactate dehydrogenase are also observed in megaloblastic anaemia, haematologic malignancies, infarction of various organs, and skeletal muscle disorders. Thus increased lactate dehydrogenase alone is not a reliable marker of haemolysis.

Intravascular haemolysis causes release of haemoglobin in circulation. Free haemoglobin combines with haptoglobin in plasma and this complex is then cleared from the circulation by hepatocytes. This causes reduction in the level of plasma haptoglobin. Low plasma haptoglobin levels also occur in extravascular haemolysis,

Figure 2.8: Mechanism of extravascular haemolysis in macrophages of reticuloendothelial system with formation of bilirubin

megaloblastic anaemia, and liver diseases. Being an acute phase reactant, plasma haptoglobin rises in inflammatory and neoplastic diseases.

Free haemoglobin (haemoglobinaemia) appears in circulation once the plasma haptoglobin dis appears. Free haem can bind albumin, leading to the formation of methaemalbumin (methaemal buminaemia). Methaemalbumin can be detected by Schumm’s test (detection of distinctive absorption band of methaemalbumin at 558 nm on spectrophotometry). Free haemoglobin is also excreted by the kidneys resulting in haemoglobinuria. Benzidine or orthotoluidine test can be used for detection of haemoglobin in urine. Some quantity of haemoglobin in glomerular filtrate is absorbed by renal tubular epithelial cells and stored as ferritin or haemosiderin. Shedding of such cells in urine results in haemosiderinuria, which can be demonstrated by iron stain.

Figure 2.9: Intravascular haemolysis

Table 2.7: Comparison of extravascular and intravascular haemolysis Parameter Intravascular haemolysis Extravascular haemolysis 1. Site of haemolysis Within circulation Macrophages of spleen, liver, bone

marrow, etc.

2. Causes Blackwater fever, incompatible

blood transfusion, PNH, PCH Haemoglobinopathies, hereditary haemolytic anaemias, autoimmune haemolytic anaemia

3. Splenomegaly Absent Present

4. Reticulocyte count Increased Increased

5. Indirect serum bilirubin Increased Increased

6. Plasma haemoglobin Markedly increased Mild to moderately-increased

7. Haemoglobin in urine Present Absent

8. Haemosiderin in urine Present Absent

9. Methaemalbumin (Schumm’s test) Positive Negative

10.Serum haptoglobin Decreased Decreased

11. Serum LDH Increased Increased

PNH: Paroxysmal nocturnal haemoglobinuria; PCH: Paroxysmal cold haemoglobinuria

etic activity is associated with reticulo-cytosis, presence of nucleated red cells, and increased numbers of white cells and platelets in peripheral blood. Young red cells with ribosomal remnants are called as polychromatic cells on Romanowsky stained smears and as reticulocytes when stained supravitally with brilliant

cresyl blue or new methylene blue. Polychromatic cells are slightly larger than mature red cells and have a faint blue-grey tint due to the presence of residual RNA. Staining of haemoglobin with acid dyes and staining of RNA with basic dyes produces poly-chromasia. These signs of accelerated erythropoiesis are also seen in acute blood loss anaemia and recovery phase of nutritional anaemias. Features common to all haemo-lytic anaemias are presented in Box 2.7.

No single test is specific for haemolysis and therefore a combination of laboratory tests is usually obtained to document the presence of haemolysis.

Tests to determine the cause of haemolysis: Various causes of haemolytic anaemia are listed in Table 2.3. Once the presence of haemolysis is established, further work-up is guided by the clinical information and peripheral smear findings (e.g. sickled forms, spherocytes). Some of the laboratory tests used for demonstrating the cause of haemolysis are–haemoglobin electrophoresis (for abnormal haemoglobins), test for glucose -6-phosphate dehydrogenase deficiency, osmotic fragility test for hereditary spherocytosis, antiglobulin (Coombs’) test for immune haemolysis, isopropanol precipitation test for unstable haemoglobins, Ham’s test for paroxysmal nocturnal haemoglobinuria, etc. These tests are discussed in respective chapters.

Approach to diagnosis of haemolytic anaemia involves establishing the presence of haemolysis followed by determination of the cause of haemolytic anaemia (Figs 2.10 and 2.11).

Normocytic anaemias with reduced reticulo-cyte count: This type of anaemia results from hypoproliferation in the bone marrow. Peripheral blood smear shows pancytopaenia with relative predomi-nance of lymphocytes in aplastic anaemia.

Leucoerythroblastic picture is a charac-teristic feature of myelophthisic anaemia.

• Clinical: pallor, mild jaundice

• Laboratory:

a. Biochemical: Increased unconjugated serum bilirubin, increased lactate dehydrogenase, decreased or absent serum haptoglobin

b. Haematological: Increased reticulocyte count, polychromasia on blood smear, erythroid hyperplasia in bone marrow

Figure 2.10: Evaluation of haemolytic anaemia

In both these conditions, bone marrow examination is essential for diagnosis. In renal failure, anaemia of chronic disorders, and hypothyroidism, clinical manifestations and ancillary laboratory studies (e.g. renal function tests) are helpful in establishing the diagnosis.

‰ ‰ ‰ ‰

‰ BIBLIOGRAPHY

1. Colon-Otero G, Menke D, Hook CC. A practical approach to the differential diagnosis and evaluation of the adult patient with macrocytic anemia. Med Clin North Am. 1992;76: 581- 597.

2. Hermiston ML, Mentzer WC: A practical approach to the evaluation of the anemic child. Pediatr Clin N Am. 2002;49:877-91.

3. Hoffman R, Benz EJ, Shattil SJ, Furie B, Silberstein LE, McGlave P, Heslop H (Eds): Hematology.

Basic principles and practice. 5th ed. Philadelphia. Churchill Livingstone Elsevier. 2008.

4. Lindenbaum John. An approach to the ane mias. In Wyngaarden JB, Smith LH Jr, and Bennett JC (Eds.): Cecil Textbook of Medi cine. 19th ed. Philadelphia. W.B. Saunders Co. 1992.

5. Massey AC. Microcytic anemia: Differential diagnosis and management of iron defi ciency ane-mia. Med Clin North Am. 1992;76: 549 -66.

6. Tefferi A. Anemia in adults: A contemporary approach to diagnosis. Mayo Clin Proc. 2003;78:

1274-280.

Figure 2.11: A simplified approach to diagnosis of haemolytic anaemias. Abbreviations: DAT:

Direct antiglobulin test; G6PD: Glucose-6-phosphate dehydrogenase; PNH: Paroxysmal nocturnal haemoglobinuria; Hb: Haemoglobin