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HEMATOLOGY:HE THE RED CELL

TERMINOLOGY

HEMATOCRIT (Hct) Hct is the percentage blood volume which is red cells. Coulter instruments measure both the number of red cells per volume of blood, and the mean cell volume (MCV) of the red cells (i.e. size of the RBCs). Hct calculated as decimal fraction (litres RBC (red cell volume) / litre blood) { x 100 = %Hct } Normal adult range for males: 40 – 50%. Females: 35 – 45%. MEAN CELL VOLUME (MCV) Mean volume of RBC (femtolitres) = the size of the RBC. Normal adult range: 82 – 96 fl. MCV < 82 = microcytosis MCV > 96 = macrocytosis. Because evaluation of the RBC size is key to the diagnosis of an anemia, the MCV is the most important of the RBC indices. MEAN CELL HAEMOGLOBIN (MCH) Average weight (picograms) of haemoglobin per RBC. Normal range: 27 – 32 pg MCH = Hb x 10 / RBC . MEAN CELL HAEMOGLOBIN CONCENTRATION (MCHC) Concentration (g / dl) of hemoglobin in the RBC. Normal range: 32 – 36 g / dl MCHC = Hb / Hct . RED CELL DISTRIBUTION WIDTH (RDW) Indicates variation in size (measure of anisocytosis) in RBC population. Normal range: 10 – 15% (standard deviation of the MCV as percentage of the MCV).


ERYTHROPOEISIS AND NORMAL RED CELL DIFFERENTIATION All cells in the Bone Marrow are derived from a pluripotential stem cell. When differentiation is initiated by a stimulus, such as the cytokine erythropoeitin, the cell begins to direct itself towards a cell lineage and is called a progenitor cell. 1. Progenitor Cell: This cell is not recognizable as a member of the erythroid lineage but can give rise to colonies of red cells. 2. Pronormoblast: This is the earliest recognizable red cell precursor. It is a large cell with blue staining cytoplasm and a nucleus which occupies most of the cell. The nuclear chromatin is fine and several small blue nucleoli are present. 3. Normoblast: The normoblast is divided into 3 different stages: (a) Basophilic: The cell has lost its nucleoli and early clumping of chromatin is seen. The cytoplasm still stains deep blue with Romanowski; indication of high nucleic acid activity. (b) Polychromic: At this stage both the cell and nucleus are smaller. Large clumps of nuclear chromatin are seen. The color of the cytoplasm varies from gray-purple to pink. (c ) Orthochromic: The cell is smaller still and the nucleus has shrunk. The nucleus is pink. 4. Reticulocyte: The cell is now almost mature. The nucleus has been extruded but the cell still contains RNA remnants in its cytoplasm. When staining with supravital stains these remnants can be seen. Still in bone marrow. (increased delivery into circulation in anemia) 5. Erythrocyte: The normal mature red cell is described as being normocytic and normochromic. XS around 7.2 micron (about the size of the nucleus of a small lymphocyte). The cell is bi-concave in shape and therefore stains palely in the center. Normal cells can have slight variations in shape and color. RED CELL MORPHOLOGY Describes the appearance of the red cell. It forms a very important part of the slide examination. In disease a variety of abnormalities can occur in red cell morphology. These are usually caused by: 1. Abnormal erythropoiesis 2. Inadequate hemoglobin formation 3. Damage to the red cell after leaving the bone marrow 4. Attempts by the bone marrow to compensate for the anemia. ABNORMALITIES IN SIZE: ●

Anisocytosis: Describes a marked variation in size. It is non-specific, appearing in many anemias and leukemia. The MCV is often normal (size differences to both sides balance out). Microcytosis: Indicates red cells which are decreased in size (<6.4 micron). They can be formed either in the marrow or may be due to fragmentation in the circulation. They usually contain less hemoglobin and are often associated with a low MCV. Most often seen in iron deficiency anemia. Macrocytosis: Indicates an increase in size (>8 micron). They are the result of abnormal hemopoeisis and are often seen in megaloblastic anemia (B12 and folate deficiency). Also detected in pernicious anemia and liver disease. Reticulocytes seen are usually larger than normal.

ABNORMALITIES IN SHAPE: ●

Poikilocytosis: This term indicates a variation in shape. It can result from abnormal


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erythropoeisis or damage to the red cells in circulation. It is seen in a variety of anemias and other conditions. Tear drop cells: Often seen in conditions where the bone marrow is replaced by nonhaemopoeitic tissue. E.g. myelofibrosis and malignant infiltrates. They are also seen in iron deficiency, thalassemia and are often associated with the pitting function of the spleen. Spherocytes: These cells have lost their bi-concavity and thus their centre pallor. They usually have a smaller diameter compared to their volume and stain very densely. Due to the loss of surface area they usually have a short survival. Spherocytes are caused by abnormal membrane proteins, lipid loss and excessive Na+ flux. They are often seen in hereditary spherocytosis and post splenectomy. They can also be seen in a number of hemolytic anemias. Ovalocytes / elliptocytes: They are oval in shape and appear in a variety of disorders. The most common is hereditary ovalocytosis, but also found in thalassemia, sickle cell anemia, iron deficiency and megaloblastic anemia. In iron deficiency these cells could be cigar shaped or pencil cells. They are caused by the inability of the red cell to return to normal after being deformed by the shear stress in the microcirculation. Target cells: Cells show an area of central staining. Caused by an increase in surface area without change in volume. They are seen in thalassemia, post splenectomy and liver disease. In liver disease it is thought they are caused by an increase in cholesterol and lecithin in the cell membrane. Sickle cells: Resemble crescents. They appear when HbS is present in high concentrations. Often associated with deoxegenation of the blood and the condition is reversable. The sickles are formed by the precipitation of polymerized hemoglobin. Fragmentation: Formation of a variety of shapes, resulting from severe mechanical or physical stress and are often seen in disorders like DIC and in patients with mechanical heart valves (microangiopathic anemias). They are also observed in burns patients. Stomatocytes: This term describes a red cell with a slit-like central pallor. They can be induced by reducing the pH and may be caused by defects of the Na+ - K+ pump. They are seen in hereditary spherocytosis, stomatocytosis and liver disease caused by alcohol abuse. Burr cells: These cells have blunt projections. They are thought to be damaged or fragmented cells and are seen in uremic patients, cancer and microangiopathic anemia. Acanthocytes: Cells which are described as 'thorny'. They are due to abnormal lipid content in the membrane. Can be inherited, and are seen in splenectomised patients and chronic liver disease. Crenation: These cells have puckered out edges. Generally have no clinical significance but rather are artefacts produced by drying of the slide and long storage of the blood. They can however occur in patients who have an electrolyte imbalance. Bite cells: When aggregates of hemoglobin form in red cells (e.g. Heinz bodies), as they pass through the spleen mononuclear phagocytes remove the aggregates leaving cells that look like a bite was taken out of them. They are seen in patients with unstable hemoglobins, G6PD deficiency and drug induced hemolytic anemias. Helmet cells: Cells shaped like a helmet. They are derived from repeated mechanical trauma and found in conditions like DIC and hemolytic anemia.

VARIATIONS IN COLOR: ●

Hypochromasia: This term indicates cells which have pale staining. This is due to thin cells and diminished hemoglobin formation. It is often associated with microcytosis and is seen in iron deficiency, thalassemia and sideroblastic anemia. Polychromasia: Indicates varying shades of staining. This is due to an increase in cells which contain RNA and take up the basic (i.e. blue) stain. They are usually immature red cells such as reticulocytes. This is most often seen in a variety of anemias such as hemolytic


anemias, megaloblastic, iron deficiency (especially after treatment) and in bleeding conditions, where bone marrow has to compensate for lack of properly functioning red cells in circulation by releasing more at a faster pace, resulting in many immature cells in circulation. Dimorphism: This term indicates the presence of two different staining red cell populations. There are 4 conditions where this can occur: 1. Iron deficiency after treatment: new normocytic / normochromic cells and the hypochromic / microcytic cells. 2. Hypochromic anemia after a transfusion: normochromic and hypochromic cells. 3. Sideroblastic anemia: Picture is variable. 4. Mixed deficiency: Large normochromic cells and microcytic / hypochromic cells.

OTHER ABNORMALITIES: ● ●

Rouleaux: Rouleaux formation when red cells arranged in stacks like coins. It is often seen when there is an increase in plasma protein concentrations such as in Myeloma. Agglutination: Describes the clumping of red cells. Seen in autoimmune hemolytic anemias.

RED CELL INCLUSION BODIES ●

Howell Jolly Bodies: These are small, well-defined spherical bodies, usually acentrically situated in the red cell. They stain deep purple and vary in size. It is generally accepted that they represent chromosomes or nuclear remnants. Since they contain DNA, they give a positive Feulgen reaction. Howell Jolly bodies are found after splenectomy or when splenic function is diminished. May also be seen in the presence of a normal spleen, when there is erythroid hyperplasia or dyerythropoeisis. Pappenheimer Bodies: Small, dense blue granules which appear at the periphery of reticulocytes or erythrocytes. They stain blue with Wrights stain and giemsa stain. They can also be demonstrated using the Perl's Prussian Blue reaction which is used to demonstrate iron. Pappenheimer bodies consist of ferric iron complexed with protein. They usually occur when splenic function is absent, e.g. post splenectomy. Basophilic stippling / Punctuate Basophilia: Appear as rounded granules which are evenly distributed throughout the red cell. They stain blue-black color and consist of aggregated ribosomes which do not contain iron. It is important to note that slow-drying of blood films and subsequent staining may cause ribosomes to aggregate; so this could be an artefact. They are demonstrated by romanowski stains. Basophilic stippling is seen in certain conditions such as thalassemia and lead poisoning, so does have diagnostic significance. In Pyrimidine-5-nucleotidase deficiency, an inherited hemolytic anemia, basophilic stippling is a notable feature. It is interesting to note that the stippling seen in lead poisoning is related to the deficiency of the same enzyme. Siderotic Granules: Siderocytes are red cells containing aggregates of non-heme iron which can only be demonstrated by the Prussian Blue stain or by electron microscopy. Usually found in normoblasts in the bone marrow – then called sideroblasts; and in reticulocytes and erythrocytes – termed siderocytes. Siderotic granules in reticulocytes usually disappear as the red cell matures, whereas siderotic granules found in the red cell are located in the mitochondria and are removed in the spleen. They are often seen in anemias like sideroblastic anemia where they form a ring around the nucleus of the normoblast and are called ringed sideroblasts. Siderocytes are seen in decreased splenic function. Heinz Bodies: Usually single round structures, situated at the cell margin. They cannot be


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seen using normal romanowski stains, but are demonstrated with supravital stains such as methyl violet and in particular methyl green. They consist of denatured, precipitated hemoglobin bound to the red cell membrane by disulphide bonds. They are produced through oxidative stress caused by drugs or chemicals in patients with inherited Glucose-6Phosphate Dehydrogenase (G6PD) deficiency. The number of heinz bodies depends on the nature of the hemoglobin defect and splenic function. They sometimes occur in premature babies with hemolytic anemia. Hemoglobin H: These inclusions appear as multiple evenly distributed blue staining bodies giving a 'golf ball' appearance to the red cell. They are best demonstrated using the supravital stain Brilliant cresyl blue. Hb H inclusions are caused by the precipitation of beta globin chains in the absence of alpha chains as seen in alpha thalassemia. Cabot Rings: Thread-like inclusions which appear in erythrocytes as rings, figure-8 or other shapes. They stain blue with romanowski dyes. They are thought to be remnants of the mitotic spindle since they give a positive Feulgen reaction for DNA. They may also contain histone protein and iron. Cabot rings are rare, but are seen in megaloblastic anemias.

Summary: Inclusion

Nature

Demonstration

Howell-Jolly bodies

DNA (nuclear remnants)

Romanowski and Feulgen stain

Pappenheimer bodies

Ferric Fe (+++) with protein

Romanowski and Prussian blue

Basophilic stippling

Ribonucleoprotein (aggregated ribosomes)

Romanowski

Protozoa

Malaria, Babesia spp

Romanowski

Micro-organisms

Bartonella bacilliformis

Romanowski

Heinz bodies

Denatured hemoglobin

Supravital staining

Hb H inclusions

Precipitated Hb H

Extended supravital staining

Siderotic granules

Fe+++ in ferritin or mitochondria

Prussian-blue reaction

Reticulocyte inclusions

Ribonucleoprotein (aggregated ribosomes)

Supravital staining


ANEMIA

Anemia, as such, is not a diagnosis but a symptom of an underlying disorder. Anemia occurs when the hemoglobin concentration drops below normal limits: Men: < 14 g/dl Women: < 12 g/dl Definition of Anemia: A reduction in the oxygen carrying capacity due to a lower hemoglobin concentration than is usual for that patient. Exceptions: 1.Normal hemoglobin but altered oxygen affinity due to 2,3-DPG deficiency. 2.Normal hemoglobin due to adequate compensation by the bone marrow. IRON DEFICIENCY ANEMIA Pathogenesis: Iron is required for normal biosynthesis of heme and thus hemoglobin. In iron deficiency: Reticuloendothelial stores are depleted. This limits erythropoeisis: The production of hemoglobin is decreased and hypochromic microcytic red cells result. Causes: Diet: Rarely the sole cause. Often seen in extreme poverty, religious diets and vegetarianism. Increased requirements: Infancy: increased demands for growth. Menstrual loss: deficiency can occur if > 80 ml blood is lost. Pregnancy: demand increases in 3rd and 4th trimester. Blood loss: Often the most common cause. Loss can occur from genital or gastrointestinal tract (e.g. chronic ulcer). Malabsorption: May occur after gastrectomy as gastric acid enhances absorption. Dietary iron is poorly absorbed in diseases like coeliac disease (gluten intolerance). Three Stages of Iron Deficiency: (1) Depletion of the body's iron stores: No anemia has yet developed. No clinical signs have appeared. The first stage is recognised by: â&#x2014;? Absence of stainable iron in the macrophages of the bone marrow. â&#x2014;? Low serum ferritin level: Ferritin present in the serum is either secreted or has leaked from the macrophages. It's function is not clear, but it provides a way of determining iron stores.


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Normal range: Women 30 – 180 mcg Men 28 – 220 mcg . Increased Total iron binding capacity: Plasma iron circulates bound to the protein, transferrin. Transferrin is not measured directly, but indirectly as the amount of iron a serum sample can bind. This is called the TIBC. TIBC rises as iron stores fall. Normal range: 75 – 100 mcg/ dl . (2) The second stage of iron deficiency is iron deficient erythropoeisis: The patient is still not anemic. Clinically: Patient may show impaired exercise tolerance Reduced learning capabilities. The stage is recognised by: Slightly reduced MCV Elevated erythrocytic protoporphyrin (heme-precursor – iron needed to form heme). This can be determined by a simple fluorescent technique. Normal range for protoporphyrin: < 35 mcg/ dl . (3) The third stage is frank Iron deficiency. All the above features are present. In addition: Hb is less than normal Low: MCV, red cell count, MCH, MCHC.

Clinical features: Breathlessness, pallor, fatigue, headaches, retinal hemorrhages, craving for unusual foods, occasional enlarged spleen. Peripheral Blood: Microcytic / hypochromic red cells. Anisocytosis Poikilocytosis: oval and pencil cells, tear drops, occasional target cells Basophilic stippling in severe anemia. Bone Marrow: Not routinely done in iron deficiency anemia. Erythroblasts: ragged, vacuolated cytoplasm and pyknotic nuclei. Smaller than normal macrophages with no iron. Other: Retic count: usually low for the amount of anemia, but can be raised in early stages. Increase after treatment has commenced. Serum iron: low TIBC: raised Treatment: Oral or parenteral iron. Response measured by increase in retics and rise in Hb levels. MEGALOBLASTIC ANEMIA Megaloblastic anemia is usually caused by either: vitamin B12 deficiency or folate deficiency. Pathogenesis: Thymidylate is a DNA nucleotide. It is formed via two pathways: (1) The Salvage pathway: Thymidylate is formed from the breakdown of old DNA. This alone


cannot generate enough thymidine. (2) Synthesis of new thymidine from uracil. Folate is involved in this pathway. Vitamin B12 (cobalamin) also plays a role by converting homocysteine to methionine. Vitamin B12 deficiency impairs DNA synthesis in two ways: 1. Trapping of folate as methyl folate. 2. Insufficient supply of methionine: methionine is also a major source of intracellular methyl tetra hydro folate. Thus, both vit. B12 and folate are involved in the synthesis of DNA. The defect in DNA synthesis will lead to abnormal hemopoeisis. This can result in a drop in Hb to as low as 4.0 g/ dl . Causes of vit. B12 deficiency: (1) Insufficient intake (2) Malabsorption: Lack of intrinsic factor, due to: failure to secrete I.F. e.g. in gastric atrophy, pernicious anemia, surgical removal of part of stomach. (3) Abnormalities of the ileum: Vit. B12 cannot be transported across membrane, due to: damage to ileum, surgery, tapeworm, drugs, congenital (rare). (4)Transcobalamin deficiency, nitrous oxide.(inadequate plasma transport).

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Causes of Folate deficiency: (1) Diet: Most common cause: Alcoholics Low income groups over cooking (2)Increased requirements: Hemolytic anemia Malignancy (increased DNA synthesis) Pregnancy Dialysis (3)Abnormal absorption: Mucosal disease of the jejenum Drugs

Clinical features: Anemia often detected before clinical symptoms appear due to the increased MCV. Main symptoms: weakness, tiredness, shortness of breath, angina, heart failure, weight loss, glossitis, jaundice, occasional enlarged spleen. Vit. B12 deficiency: occasional neurologic symptoms (role in myelin) Laboratory findings: Full Blood Count: Hb: decreased MCV: increased (> 95 fl) WBC: often low Platelets: often low NB: Pancytopaenia (low RBC, WBC and platelets) is a feature of megaloblastic anemia. Peripheral Blood smear: RBC: macrocytes (oval macrocytes) anisicytosis poikilocytosis: tear drops and oval macrocytes nucleated red cells basophilic stippling Indicators of abnormal red cell development


howell jolly bodies Indicators of abnormal red cell development cabot rings Neutrophils: often hypersegmented (> 5 lobes in nucleus) Platelets: often decreased Reticulocytes: normal or low as abnormal new cells are destroyed before leaving the marrow. Bone Marrow: Erythropoeisis: abnormal with predominance of megaloblasts. They have primitive nuclei, even though the cytoplasm is mature. Clover leaf nuclei are often seen. Granulocytes: giant metamyelocytes and band cells. Megakaryocytes: abnormal with increased nuclei. Vit. B12 normal range: 180 – 900 mcg/ l Serum folate normal range: 3 – 20 mcg/ l If both are reduced: ? Combined deficiency. If both are normal: ? Myelodysplasia Red cell folate often a better assessment of body stores. Normal range: 160 – 700 mcg/ l . Schillings test done to determine vit. B12 absorption. If abnormal, repeated adding intrinsic factor to the dose of vit. B12. Other tests: The deoxyuridine suppression test. Response to replacement therapy. Therapy: Treat with vit. B12 and folate. In severe anemia transfusion may be needed. Reticulocytosis occurs within 5 – 7 days of therapy. PERNICIOUS ANEMIA (a type of Megaloblastic anemia) Pathogenesis: Severe gastric atrophy with failure to secrete intrinsic factor. Most patients have antibodies to both IF producing parietal cells and to IF. → impaired B12 absorption. Clinical features: Usually occurs in elderly people Weakness, fatigue, gastrointestinal symptoms. Laboratory Findings: Peripheral blood and bone marrow show megaloblastic changes. Vit. B12 levels are decreased. Schillings test: decreased; corrected with addition of intrinsic factor. Antibodies: 90 % of patients have antibodies to parietal cells and intrinsic factor. ? Autoimmune disorder. Treatment: Vit. B12 indefinitely Note: Folate especially also vital in prevention of neural tube defects in foetus, and lowering incidence of cardiovascular disease, through lowering of homocysteine levels.


Summary: RBC in Anemia: –

Hypochromic / microcytic anemias Iron deficiency Disorders of globin synthesis: thalassemia syndromes Disorders of heme synthesis: sideroblastic anemias

Macrocytic anemias Megaloblastic bone marrow: Vit. B12 deficiency Folate deficiency Disorders of DNA synthesis – hereditary, acquired Non-megaloblastic bone marrow: Accelerated erythropoeisis with polychromasia, normoblasts I in the peripheral blood. : Increased cell membrane surface area – e.g. hepatic disease

Normochromic / normocytic anemias Recent blood loss Hemolytic disorders Hypoplastic anemias Infiltrated bone marrow: May see leucoerythroblastic features in peripheral blood smear: tear drop cells, normoblasts, primitive white cells

HEMOLYTIC ANEMIA AND THE THALASSEMIAS HEMOLYSIS: RBC

GLOBIN

IRON

AMINO ACIDS

PROTOPORPHYRIN BILIRUBIN

Binds to Transferrin – Reused UNCONJUGATED BILI

LIVER

UROBILINOGEN (Urine)

STERCOBILINOGEN (Feces)


THE HEMOLYTIC ANEMIAS Definition: A hemolytic anemia develops when there is a reduction in the life span of the red cell and the bone marrow is unable to compensate for this loss. TESTS TO CONFIRM HEMOLYSIS General findings: Retic Count: Increased Heinz bodies may also be seen Blood Smear: Polychromasia and nucleated red cells May also see (depending on type of hemolytic anemia) sherocytes, fragments, agglutination, sickle cells, bite cells, target cells. Extravascular hemolysis This is due to red cell destruction which occurs outside the circulation. Sites may be: Spleen, Liver and Bone Marrow. Tests: Unconjugated bilirubin: increased Urobilinogen: increased Stercobilinogen: increased Intravascular hemolysis This is due to the breakdown of red cells within the circulation. The globin chains will bind to a plasma molecule called haptoglobin. The complex is removed by the liver. Therefore, In plasma: Haptoglobin levels will be extremely low (Normal: 150mg/ dl) Plasma Hb will be increased Methaemalbumin and MetHb will be increased LDH levels will be increased. In urine: Hemoglobinuria and hemosiderin ABNORMALITIES DUE TO MEMBRANE DEFECTS: â&#x2014;?

HEREDITARY SPHEROCYTOSIS

Pathogenesis: Autosomal dominant Cells have reduced spectrin content of the membrane skeleton This causes loss of membrane integrity and therefore, sphere formation The spherocytes are eventually phagocytosed by macrophages in the liver and spleen â&#x20AC;&#x201C; i.e. Extravascular Hemolysis . Clinical features: Variable anemia Jaundice


Increased excretion of bilirubin and therefore increased incidence of gall stones. Enlarged spleen Leg ulcers (unknown cause). Laboratory findings: Full Blood Count: variable anemia, increased MCHC Blood smear: increased spherocytes increase in polychromasia and reticulocytes. Other tests: increased osmotic fragility mildly elevated serum bilirubin negative coombs (i.e. not immune disorder) increased urobilinogen. Crises: The bone marrow usually maintains a constant Hb. However often after stress or viral infections the bone marrow cannot cope and anemia occurs. Common cause: Parvovirus. Therapy: Splenectomy often rectifies anemia. ●

HEREDITARY ELLIPTOCYTOSIS / OVALOCYTOSIS

Very rare condition. Pathogenesis: Caused by defect in the membrane protein spectrin. The cells have decreased deformability They are eventually removed by the spleen – thus, extravascular hemolysis. Three forms: 1. Asymptomatic: Ovalocytes are seen on the smear but little or no hemolysis is experienced. 2. Combination of spherocytosis and ovalocytosis: The anemia is usually mild. 3. Combination of ovalocytosis, spherocytosis and fragmentation: Very rare disorder. Moderate anemia. ●

OTHER HEREDITARY MEMBRANE DISORDERS

Pyropoikilocytosis Stomatocytosis ●

Both conditions very rare.

ACQUIRED MEMBRANE DEFECTS

PAROXYSMAL NOCTURNAL HEMOGLOBINURIA Pathogenesis: Cells have intrinsic membrane abnormality which triggers the complement cascade causing lysis of the red cell. This is intravascular hemolysis . The abnormality occurs at the pluripotential stem cell in the bone marrow, due to a gene mutation. It is a clonal disorder, but not a malignancy, but in some patients it can go on to develop Acute Leukemia. The condition is caused by a reduction in DAF or CD55 (regulates complement activation) and


another surface protein CD59. The underlying reason is the GP1 anchor on the membrane is missing due to mutation of the PIG A gene. Clinical Features: Often occurs after drug induced bone marrow aplasia. Fatigue There is usually increased hemolysis at night due to a fall in pH during sleep. Morning urine is often discolored, hence the name of the condition. Pallor, jaundice . Laboratory Features: Anemia with low Hb Increased reticulocytes Low white cell count and platelets, i.e. pancytopenia . Peripheral Blood smear: No abnormalities Increased polychromasia Other tests: Increased plasma Hb Increased methemoglobin Increased plasma bilirubin Increased hemosiderin Coffee colored urine after hemolysis Sucrose lysis test (concentration gradient test) and Hams acid test for increased lysis due to complement: Positive . Complications: Thrombosis, Iron deficiency, and development of Leukemia. Therapy: Bone marrow transplant Iron therapy Corticosteroids Anticoagulant therapy after thrombosis. HEMOLYSIS CAUSED BY ANTIBODIES Two main groups of antibodies: IgG and IgM . The Coombs test investigates antibody-involvement in the destruction of red cells. Direct Coombs: ? Antibodies bound to cell surface. Indirect Coombs: ? Antibodies in the serum. Alloantibodies: ABO Reactions Patients receiving incompatible blood. Hemolysis occurs due to antibodies reacting with the donor red cells. â&#x20AC;&#x201C;

HDN Pathogenesis: â&#x20AC;&#x201C;


Hemolytic Disease of the Newborn usually due to Rh incompatibility. Rh-neg. Mother sensitised to Rh-factor (D-antigen), e.g. from previous pregnancy. Her anti-D (usually IgG) crosses the placenta → hemolysis of Rh-pos. baby's red cells. See also: http://www.scribd.com/doc/12600118/Blood-Transfusion-Notes Pages 11, 15 and 16. See also: http://www.scribd.com/doc/11855527/immunology-notes Treatment: See Blood Transfusion Notes. Autoantibodies: Warm antibodies Usually IgG Can be idiopathic (no apparent cause) Usually secondary to an underlying disease. –

Pathogenesis: The red cells are coated with the antibody. They are then removed by the spleen, liver and bone marrow. Macrophages engulf pieces of the cell, resulting in the damaged cells forming spherocytes. They can also fix complement resulting in hemolysis. Clinical features: Weakness, malaise, fever, jaundice, splenomegaly . Laboratory Findings: White cell count and platelets: Normal or slightly raised. Smear: Polychromasia Nucleated red cells Spherocytes Increased reticulocytes . Coombs: Positive Serum bilirubin: Increased Treatment: Transfusion Steroids, immunosuppressives Splenectomy Cold antibodies Usually IgM. Do not normally react with red cells in the body unless: In case of certain infections like Cytomegalovirus and mycoplasma. Cold agglutination disease: seen in older patients often with underlying lymphoma. –

Pathogenesis: As cells pass through cooler peripheral areas, IgM attaches to red cell surface and activates complement. The red cells are usually removed by the spleen and liver.


Laboratory findings: Agglutination is seen on the smear. Polychromasia and spherocytes. Cold agglutination test: Positive Coombs test positive. Cell count: High MCHC due to agglutination – incubate sample at 37 deg.Celsius to remove cold antibodies. HEMOLYSIS DUE TO ABNORMAL RED CELL METABOLISM: These anemias are caused by deficiencies of enzymes found in the glycolytic pathway and the hexose monophosphate shunt. ●

PYRUVATE KINASE DEFICIENCY

Pathogenesis: This is the most common enzyme deficiency of the glycolytic pathway. The others are extremely rare. Deficiency results in the failure of the red cell to produce ATP. Hemolysis occurs because the sodium pump fails to control Na+ influx. This leads to osmotic lysis. Clinical symptoms: Inherited disorder Anemia and jaundice are present from birth. The anemia worsens during an infection. Often find enlarged spleen. Laboratory findings: This disorder does not have any distinctive feature. Increased serum bilirubin, increased polychromasia and increased reticulocytes point to possible hemolysis. No spherocytes Decreased pyruvate kinase assay is NB. Therapy: Transfusions and splenectomy. ●

G6PD DEFICIENCY

G6PD is the most common enzyme deficiency. Pathogenesis: G6PD is part of the hexose monophosphate shunt which is the oxidation-reduction system of the cell. It is vital to detoxify oxidising agents entering the red cell. In G6PD deficiency the system cannot function and there are two major effects: 1. Oxidation of globin chains to form Heinz bodies. 2. A failure to reduce methemoglobin. The Heinz bodies are removed by the spleen. Clinical features: Sex linked inheritance. Usually affects males. Seen in Malaria affected areas -- ? protection.


Hemolysis usually occurs after exposure to a drug or an infection, e.g. anti malaria drugs, sulphonamides, analgesics, eating Fava beans. This precipitates a hemolytic attack with jaundice, weakness and dark urine. Laboratory diagnosis: Anemia (low Hb) Polychromasia with increased retics. Heinz bodies and consequent bite cells. Increased bilirubin and urobilinogen. Decreased G6PD screen. DRUG INDUCED HEMOLYSIS: Antibodies can bind to a red cell / drug complex (e.g. Penicillin). Complex removed by spleen. RED CELL FRAGMENTATION OR MICROANGIOPATHIC ANEMIA: The anemia is caused by direct damage to the red cell. Artificial heart valves Passing through fibrin strands caused by DIC Anemia Malignancies secreting mucin Thrombotic thrombocytopenic purpura (TTP). The main feature of the smear is the presence of Fragmentation. â&#x2014;?

HEMOLYTIC UREMIC SYNDROME

A specific syndrome involving microangiopathic anemia. Need to distinguish diagnostically from DIC. HUS was first described in children where it was associated with an intravascular hemolytic anemia and renal failure. Pathogenesis: The toxins and verotoxins produced by the bacteria commonly associated with this disorder (E.coli, Shigella dysenteriae, Salmonella typhi and Streptococcus pneumonia) cause vascular endothelial damage to the glomerular capillaries, renal arterioles and other vessels. Following this vascular injury: Factor VIII / Von Willebrand Factor is released. Platelets become activated and are also damaged by the various toxins. Red cells are damaged by the bacterial toxins, and also by fibrin strands that form intravascularly. Clinical features: HUS is characterised by: Renal microangiopathy involving arterioles and glomerular capillaries. Platelet destruction â&#x2020;&#x2019; Thrombocytopenia Anemia . Different forms of HUS have been identified: 1.Classic form: Occurs in healthy infants. Symptoms include: vomiting, blood diarrhoea, anemia, renal failure. Multisystem involvement can occur.


Verotoxin producing E.coli is usually associated with this form. 2.Post infectious form: Associated with infections of Shigella dyseteriae, Salmonella typhi and Streptococcus pneumoniae. 3.Hereditary forms Autosomal recessive. 4.Sporadically occurring adult forms: Usually seen in association with pregnancy, oral contraception and cytotoxic agents. Laboratory findings: Anemia: low Hb Fragmented red cells, burr cells, some microspherocytes Raised retic count Increased hemosiderin. Leucocytosis with increased neutrophils Thrombocytopenia (low platelets with reduced lifespan) Can have abnormal INR and APTT but usually the clotting factors are normal. Chemistry: Blood urea and serum creatinine levels are usually increased. Treatment: Treat acute renal failure: dialysis Packed cell transfusions Fresh frozen plasma combined with plasmapheresis Treat infection . THE HEMOGLOBINOPATHIES: Normal hemoglobin consists of 2 globin molecules, forming a tetramer of 2 alpha and 2 beta chains. This is Hb A . Adult hemoglobin is made up of the following types: NAME

GLOBIN CHAINS

Hb A Hb Ac Ηb A2 Hb F (fetal Hb)

α2β2 α2β2 α2δ2 α2γ2

% OF TOTAL HB 95 < 3.5 < 3.5 < 1.0

Many abnormal hemoglobins have been discovered, e.g. Hb S in Sickle cell anemia. Abnormalities are usually caused by amino acid substitutions / deletions / insertions in the globin chain. When the substitution causes clinical symptoms the patient has a hemoglobinopathy. In cases where there is not amino acid substitution, but reduced synthesis the patient has a thalassemia. ●

SICKLE CELL ANEMIA

Seen in 5 – 20% of populations in West and Central Africa. Also in the Mediterranean, Middle East and India. These areas correspond to Malaria Areas and it is thought that Sickle cell anemia affords some sort of protection from malaria (like G6PD deficiency).


Pathogenesis: Hb S is formed by the substitution of glutamic acid with valine at position 6 of the β globin chain. Red cells wich contain increased amounts of Hb S form sickle cells when deoxygenation takes place. This is caused by the polymerisation and gelation of the Hb molecule. Sickle cells have a shortened lifespan. They are removed by the spleen and liver. SICKLE CELL TRAIT These patients have just one abnormal β chain. They have no clinical disease. Laboratory Diagnosis: Sickle cells will form if the cells are incubated with Sodium Metabisulphite which deoxigenates the blood. Hb Electrophoresis: Hb A: 60 – 70% Hb S: 30 – 40% Hb F: normal . SICKLE CELL DISEASE These patients are homozygous for Hb S, i.e. both β chains are abnormal. Clinical features: Chronic hemolytic anemia from birth. Hepatosplenomegaly Leg ulcers . Sickle cell crisis: Occurs after infection or other identifiable cause. Sickle cells increase in circulation. Patient suffers extreme pain. Cells can get caught up in the spleen. Aplastic crises: The bone marrow is unable to respond to the anemia and does not produce any new reticulocytes. Usually seen after parvovirus infection, toxins or drugs. Laboratory: FBC: low Hb normal or slightly increased MCV increased white cells increased platelets. Blood Smear: increased sickle cells (especially during crises) polychromasia Howell Jolly Bodies increased retics. Hb Electrophoresis: Hb S: 75 – 95% remainder is Hb F Serum bilirubin: slightly increased X-ray: widening of marrow space. Treatment:


Prevention of crises. Transfusion in aplastic crises. HB S CAN BE FOUND IN COMBINATION WITH OTHER ABNORMAL HB Hb S / Hb C: Most common. Results in similar conditions to sickle cell disease. Hb S / Hb D and Hb S / Hb E: This results in a mild condition. Hb S / α thalassemia: This combination increases the life span of sickle cell patients. MCV: low Hb S / β thalassemia: Increases expression of the Hb S gene. No normal β chains are produced. Severity of disease similar to sickle cell disease. MCV: low Hb Electrophoresis: mostly Hb S. ●

HEMOGLOBIN C AND E

Both disorders produce mild anemias. Hypochromic / microcytic red cells. Increased target cells. ●

UNSTABLE HEMOGLOBINS

Pathogenesis: The unstable Hb denature and form Heinz Bodies. The cells are removed by the spleen causing a hemolytic anemia. Patients have an enlarged spleen. Laboratory findings: Polychromasia and increased retics. Heinz Bodies Bite cells Positive for unstable hemoglobins (precipitate when lysed red cells are incubated with isopropanol). ●

HEMOGLOBIN M

Methemoglobin. Extremely stable form of Hb . Formed when ferrous (++) iron has been oxidised to ferric (+++) iron. Met-Hb cannot carry oxygen. Babies with G6PD deficiency are unable to reduce ferric to ferrous Fe, resulting in Met-Hb formation. ● ALTERED AFFINITY HEMOGLOBINS Have increased affinity for oxygen; thus do not release it to the tissues.


THALASSEMIA: Results from abnormal globin chain synthesis. ●

α THALASSEMIA

Four functional α chain genes inherited: two from each parent. Normal: (αα, αα) 1. Deletion of one of these genes produces no clinical symptoms. (αα, α−) This is a silent carrier. 2. Deletion of 2 genes: (α−, α−) or (αα, −−) . Clinical: Minimal anemia. Deletion forms of α thal. may provide protection against Malaria (P. falciparum and vivax). Laboratory findings: Slightly reduced Hb Slightly raised red cell count (clue that it is not a iron deficiency anemia) Low MCV Microcytosis and hypochromia Target cells Electrophoresis: normal A2 and F. The diagnosis is made by excusion. 3. Hemoglobin H disease: Deletion of 3 genes. (α−, − −) . Reduced α chain synthesis, with accompanied increase in β chain synthesis. The β chains form tetramers creating the abnormal Hb H. Abnormal HbF is also formed with γ chain tetramers. Clinical: Lifelong moderate anemia. Laboratory findings: FBC: reduced Hb reduced MCV (60 – 70 fl) Smear: microcytosis and hypochromasia target cells, ovalocytes basophilic stippling increased retics. Electrophoresis: Hb H very fast moving Hb. New Methylene Blue stain: red cell inclusions -- “golf ball”. 4. Deletion of all 4 genes (- -, - -) This disorder is incompatible with life and is called Hydrops fetalis. The abnormal foetal Hb formed is called γ4 (Hemoglobin Barts). It cannot carry oxygen.


β THALASSEMIA

Can be caused either by gene deletions or gene mutations. This can either completely suppress β chain synthesis (β 0 thal) or may impair β chain synthesis (β+ thal). There is increased α chain production which precipitates to form inclusions. Increased γ chains result in increased Hb F. 1. Heterozygote: Usually asimptomatic. FBC: decreased Hb slightly increased red cells low MCV Smear: microcytosis and hypochromasia target cells basophilic stippling HbA2 increased (4 – 6%) (determined by chromatography) HbF slightly increased. 2. Homozygote: Clinical: Severe anemia Hepatosplenomegaly and jaundice Bone changes due to expanded marrow cavity. Laboratory findings: Smear: microcytic / hypochromic red cells target cells, ovalocytes polychromasia nucleated red cells increased retics. Electrophoresis: small amount of HbA can be detected. HbA2 is increased HbF is increased (60 – 100%) (compensatory) Treatment: Repeated transfusions. Can lead to iron overload, eventual organ damage. Patient needs iron chelation therapy. Splenectomy (to relieve hemolytic anemia) Bone marrow transplant is the only cure.


THE SIDEROBLASTIC ANEMIAS: (only acquired Sideroblastic Anemia is classified under the Hemolytic anemias) Pathogenesis: The Sideroblastic anemias are diverse and vary in their pathogenesis and prognosis. All have defective heme synthesis in the red cell population which has a normal iron content. The cause is a reduction in the enzyme ALA-synthase which catalyses the first step in protoporphyrin synthesis. This results in the accumulation of coarse ferritin granules in the mitochondria. The mitochondria encircle the nucleus, the cells are called ringed sideroblasts. â&#x2014;?

HEREDITARY SIDEROBLASTIC ANEMIA

Clinical: Rare recessive disorder which is transmitted via the X-chromosome. It is therefore seen predominantly in males. Patients suffer from anemia which is prominent from the first year of life. Laboratory findings: FBC: decreased Hb decreased MCV decreased MCHC Smear: Microcytic / hypochromic red cells Often dimorphic picture present Siderocytes are seen on smear when Iron stain is done Decreased retics . Bone Marrow: Erythroid hyperplasia Ragged and vacuolated normoblasts Macrophages have adequate iron Iron stain shows many siderotic granules in normoblasts and many ringed sideroblasts Serum iron is raised. â&#x2014;?

ACQUIRED SIDEROBLASTIC ANEMIA

Primary or idiopathic: This is the same disease also called Refractory Anemia with Ringed Sideroblasts. It is classified under the Myelodysplastic Syndromes. At least 15% of the normoblasts in the bone marrow must be ringed sideroblasts. Secondary: This usually occurs as a result of drugs or toxins. Alcohol: megaloblasts and ringed sideroblasts. Anti-tuberculosis drugs: inhibit pyridoxine metabolism which is a co enzyme to ALA synthase. Chloramphenicol Lead: impairs heme synthesis at a variety of steps. Formation of ringed sideroblasts. (Basophilic stippling is another feature of lead toxicity.) Vitamin B6 deficiency. Therapy: Pyridoxine, B12 and Folate. Withdrawal of offending drug.


ANEMIA OF CHRONIC DISORDERS: This anemia is seen in patients with chronic infection or malignancy. It does not respond to iron, B12 or folate. It is caused by: An increase in red cell destruction due to stimulation of macrophage activity (e.g. due to infection). The bone marrow fails to compensate. A reason for the lack of compensation is: Iron release from the macrophages is impaired and therefore, although iron stores are normal, erythropoeisis is limited. Why? It has been shown that: Activated macrophages secrete Interleukin 1 which causes rapid fall in plasma iron. Activated neutrophils secrete lactoferrin which binds the iron. This complex is reinjested by the macrophage. Activated macrophages synthesise apoferrin which facilitates the retention of iron. Thus, it can look like an iron deficiency anemia. Laboratory features: Often normocytic / normochromic (MCV 75 – 90) Retics: normal Free protoporhyrin is increased (same as in Fe deficiency anemia) Serum ferritin is increased and TIBC reduced. Macrophages contain increased amounts of iron. Treatment: Treat primary disorder.

OTHER ANEMIAS ACUTE BLOOD LOSS ANEMIA Acute blood loss: Depletion of blood volume is important. The blood constitution remains unaltered, until plasma volume is replaced, causing dilution of the hematocrit.(e.g. IV fluid given) Chronic (gradual) blood loss like a bleeding ulcer: Body compensates for volume loss quickly but loss of red cells will affect O2 delivery to tissues. Clinical features: Depend on amount of blood loss. < 20% loss: no noticeable symptoms 30 – 40% : fall in cardiac function and onset of shock. > 40% : organ damage and often death. The body will attempt to replace the lost blood volume by increasing the plasma volume → reduced red cell mass and anemia. Laboratory features: Hematocrit : No change until plasma volume is replaced. Then it will drop. White cells: Increase after a few hours; Platelets: Dramatic increase. Erythropoeitin: Increased


Erythropoeitic response: Slow process. Progenitor cells will mature over 2 â&#x20AC;&#x201C; 3 days. By tenth day retics will peak. Retics appear earlier in response to erythropoeitin. NB: Polychromasia is seen on the smear. Therapy: Maintain blood volume to prevent shock. Blood transfusions, electrolyte solutions, saline. Iron therapy if stores depleted. APLASTIC ANEMIA AND PURE RED CELL APLASIA Patients with aplastic anemia have: pancytopenia, hypocellular bone marrow . Pathogenesis: Two mechanisms: 1. Depletion of hemopoeitic stem cells by an agent or event which kill the stem cells, e.g. radiation, chemo, benzene. 2. Suppression of proliferation of stem cells by an immune mechanism. (antibodies or T / Bcells) Causes of this condition: Genetic or congenital: Fanconis anemia. These children usually have other abnormalities like stunted growth and limb deformities. Toxins or radiation Drugs: Those that damage resting stem cells and those that affect cells during their cycle. (cytotoxic drugs) Infection: Hepatitis, Infectious mononucleosis, parvovirus. Pregnancy. Clinical findings: Weakness with cardiovascular and cerebral symptoms. Infections: low white cell count Bleeding: low platelets Pallor, petechiae. Laboratory findings: FBC: Pancytopenia: low white cells, platelets, Hb Smear: Normocytic / normochromic anemia Very low reticulocytes Few white cells and platelets. Bone Marrow: Hypocellular â&#x20AC;&#x201C; Trephine : Almost total replacement by fat spaces. The remaining cells are usually plasma cells, lymphocytes and mast cells. Treatment: Very serious condition. Bone marrow transplant only hope of cure. Androgens, immunosuppression.



HEMATOLOGY THE RED CELL