6- Hemolytic Anemias.pdf

Hemolytic Anemias Introduction to hemolytic anemias  Hemolytic anemias are defined as those anemias that result from an increase in the rate of red cell destruction.  Because of erythropoietic hyperplasia and extension of bone marrow, red cell destruction may be increased several-folds befo...

Hemolytic Anemias Introduction to hemolytic anemias  Hemolytic anemias are defined as those anemias that result from an increase in the rate of red cell destruction.  Because of erythropoietic hyperplasia and extension of bone marrow, red cell destruction may be increased several-folds before the patient becomes anemic- compensated hemolytic disease.  The normal adult marrow, after full expansion, is able to produce red cells at 5-7 times the normal rate.  It leads to a marked reticulocytosis.  Therefore, hemolytic anemia may not be seen until the red cell lifespan is marked decrease.  Erythroid hyperplasia is excessive growth of immature red blood cells Classification of Hemolytic Anemias Based on Underlying Defect  Hereditary hemolytic anemias are the result of 'intrinsic' red cell defects whereas acquired hemolytic anemias are usually the result of an 'extracorpuscular' or 'environmental' change. Difference in blood transfusions !! Intrinsic causes Hereditary (inherited) hemolytic anemia can be due to : Defects of red blood cell membrane production (as in hereditary spherocytosis and hereditary elliptocytosis) Defects in hemoglobin production (as in thalassemia, sickle-cell disease and congenital dyserythropoietic anemia) Defective red cell metabolism (as in glucose-6-phosphate dehydrogenase deficiency and pyruvate kinase deficiency) Extrinsic causes Acquired hemolytic anemia may be caused by immune-mediated causes, drugs and other miscellaneous causes. Immune-mediated causes could include transient factors as in Mycoplasma pneumoniae infection (cold agglutinin disease) or permanent factors as inautoimmune diseases like autoimmune hemolytic anemia (itself more common in diseases such as systemic lupus erythematosus, rheumatoid arthritis, Hodgkin's lymphoma, and chronic lymphocytic leukemia).  Paroxysmal nocturnal hemoglobinuria (PNH), sometimes referred to as Marchiafava- Micheli syndrome, is a rare, acquired, potentially life-threatening disease of the blood characterized by complement-induced intravascular hemolytic anemia. Any of the causes of hypersplenism (increased activity of the spleen), such as portal hypertension. Acquired hemolytic anemia is also encountered in burns and as a result of certain infections. Lead poisoning resulting from the environment causes non-immune hemolytic anemia. Runners can suffer hemolytic anemia due to "footstrike hemolysis", owing to the destruction of red blood cells in feet at foot impact. Low-grade hemolytic anemia occurs in 70% of prosthetic heart valve recipients, and severe hemolytic anemia occurs in 3%. Alloimmunity (sometimes called isoimmunity) is an immune response to nonself antigens from members of the same species  Laboratory findings The laboratory findings are conveniently divided into three groups. 1- Features of increased red cell breakdown:  Serum bilirubin raised  Urine urobilinogen increased  Fecal stercobilinogen increased 2- Features of increased red cell production:  Reticulocytosis  Bone marrow erythroid hyperplasia 3- Damaged red cells:  Morphology (e.g. spherocytes, elliptocytes)  Osmotic fragility  Red cell survival shortened  Mechanism of cell destruction There are two main mechanisms whereby red cells are destroyed in hemolytic anemia:  There may be excessive removal of red cells by cells of the spleen (extravascular destruction).  Or they may be broken down directly in the circulation in a process known as intravascular. paroxysmal nocturnal hemoglobinuria (PNH) an acquired blood cell abnormality with pr oliferation of abnormal red blood cells (PNH cells) that are readily hemolyzed by complement, and episodes of severe hemolysis and thrombosis, particularly of the hepatic veins  Hereditary Hemolytic Anemias 1- Membrane defects Hereditary spherocytosis Hereditary Elliptocytosis 2- Defective red cell metabolism Glucose-6-phosphate dehydrogenese deficiency Pyruvate kinase deficiency 3- Hereditary disorders of hemoglobin synthesis Hb.S Hb.C Hereditary Spherocytosis (HS)  Deficiencies or defects of spectrin, ankyrin (2.1), or band 3 protein cause the lipid bilayer to decouple from the underlying skeletal lattice and subsequent membrane loss in the form of microvesicles. This leads to the formation of spherocytes (hereditary spherocytosis). Horizontal interactions are parallel to the plane of the membrane. These interactions provide mechanical stability to the membrane. Defect in spectrin (α and β) and in ankyrin which leads to defective attachment of the skeleton proteins (skeletal lattice) with the lipid bilayer (vertical proteins interaction). Change the shape of RBCs from biconcave to spherocyte (less deformable). Removal of HS cells in the spleen  Findings Reticulocytosis (polychromasia) Small, dense microspherocytes MCV normal or slightly decreased (77-85fL), if reticulocytes are markedly increased, the MCV can also above 85 fL. The MCH is normal and MCHC is increased. Spherocytes are the most common erythrocytes with an increased MCHC.  Normal RBC is biconcave. RBCs in hereditary spherocytosis is spherical. Basic geometry: The shape with the smallest volume that can be occupied by a mass is a sphere. In hereditary spherocytosis, small bits of membrane are lost and the RBCs form a spherical shape and hence the volume is reduced i.e MCV is reduced. Now this is just for the spherocytes. In hereditary spherocytosis there is increased number of reticulocytes which are large in size compared to the mature RBCs. Thus the final number is a normal to low MCV. MCHC= Hemoglobin/ Hematocrit. The hemoglobin is slightly reduced. The hematocrit part is a bit complex. Its reduced but is made of spherical cells. The overall effect is a drop in hematocrit more than hemoglobin resulting in a higher MCHC.  Hereditary Spherocytosis Peripheral blood from a patient with hereditary spherocytosis shows the presence of many densely staining spherocytes (arrows) (peripheral blood, Wright-Giemsa stain, 1000* magnification).  Osmotic fragility test in HS Measures the erythrocytes resistance to hemolysis by osmotic pressure. In HS, because of their decrease surface area to volume ratio, spherocytes are unable to expand as much as normal discoid shape. Very little fluid needed to be absorbed before the cells hemolyze. Therefore, HS cells exhibit increased osmotic fragility.  Hereditary Elliptocytosis (HE) The principal defect involves horizontal membrane proteins interactions. Decrease association of spectrin dimers to form tetramers. Abnormalities of the integral proteins including glycophorin and Band 3 protein. Glycophorin C has the function of holding band 4.1 to the cell membrane.  Spur Cell Anemia  Spur cell hemolytic anemia, is a form of HEMOLYTIC ANEMIA that results secondary to severe impaired liver function or cirrhosis.  Chronic liver disease impairs the liver's ability to esterify cholesterol, causing free cholesterol to bind to the red cell membrane, increasing its surface area.  This condition also creates rough or thorny projections on the erythrocyte named acanthocytes.  Spur cell hemolytic anemia, is a form of hemolytic anemia that results secondary to severe impaired liver function or cirrhosis. Chronic liver disease impairs the liver's ability to esterify cholesterol, causing free cholesterol to bind to the red cell membrane, increasing its surface area. This condition also creates rough or thorny projections on the erythrocyte named acanthocytes. Acanthocytes  Enzymatic deficiency Glucose-6-phosphate dehydrogenase deficiency (G6PD deficiency) also known as favism (after the fava bean) is an X-linked recessive genetic condition that predisposes to Hemolysis (spontaneous destruction of RBC) and resultant Jaundice in response to a number of triggers, such as certain foods, illness, or medication. It is particularly common in people of Mediterranean and African origin. The condition is characterized by abnormally low levels of Glucose- 6-phosphate dehydrogenase; an enzyme involved in the pentose phosphate pathway that is especially important in the RBC.  Glucose-6-phosphate dehydrogenase deficiency (G6PD deficiency) also known as favism (after the fava bean) is an X-linked recessive genetic condition that predisposes to hemolysis (spontaneous destruction of red blood cells) and resultant jaundice in response to a number of triggers, such as certain foods, illness, or medication. It is particularly common in people of Mediterranean and African origin. The condition is characterized by abnormally low levels ofglucose-6- phosphate dehydrogenase, an enzyme involved in the pentose phosphate pathway that is especially important in the red blood cell. G6PD deficiency is the most common human enzyme defect. There is no specific treatment, other than avoiding known triggers. Carriers of the G6PD allele appear to be protected to some extent against malaria, and in some cases affected males have shown complete immunity to the disease. This accounts for the persistence of the allele in certain populations in that it confers a selective advantage. G6PD deficiency resulted in 4,100 deaths in 2013 and 3,400 deaths in 1990. Enzymatic deficiency  Nicotinamide adenine dinucleotide phosphate The primary results of the pathway are: The generation of reducing equivalents, in the form of NADPH, used in reductive biosynthesis reactions within cells (e.g. fatty acid synthesis). Production of ribose 5-phosphate (R5P), used in the synthesis of nucleotides and nucleic acids. Production of erythrose 4-phosphate (E4P) used in the synthesis of aromatic amino acids.  Pathophysiology G6PD is necessary for maintaining adequate levels of glutathione GSH for reducing cellular oxidants. In G6PD deficiency, hemoglobin is oxidized to methemoglobin, with further oxidation leads to its denaturation and precipitation in the form of Heinz bodies. Heinz bodies attach to the erythrocyte membrane causing cell rigidity. Heinz bodies are removed from the erythrocytes by splenic macrophage, producing BITE CELL. With progressive membrane loss, spherocytes can be formed (extravascular hemolysis, less deformable). Oxidation of membrane proteins and lipids also leads to disruption of membrane integrity. Cell membrane damage can be severe enough to cause intravascular hemolysis (hemoglobinuria and decreased haptoglobin).  Heinz bodies are formed by damage to the hemoglobin component molecules, usually through oxidant damage, or from an inherited mutation (i.e. change of an internal amino acid residue). As a result, an electron from the hemoglobin is transferred to an oxygen molecule, which creates a reactive oxygen species (ROS) that can cause severe cell damage leading to premature cell lysis. Damaged cells are cleared by macrophages in the spleen, where the precipitate and damaged membrane are removed, leading to characteristic "bite cells". The denaturing process is irreversible and the continual elimination of damaged cells leads to Heinz body anemia.  NOTES G6PD activity is highest in young RBCs and decreases as the cell ages. Normally, RBCs used only < 10% of their maximum G6PD enzyme capacity, therefore, even older RBCs normally retain enough G6PD activity to maintain enough GSH levels. Symptoms appear upon exposure to excessive oxidants. (oxidizing drugs or ingestion of fava beans-Favism).  In most G6PD, hemolysis is self limited (referring to the fact that hemolysis stops after a time even the oxidant stress continues). But in case of severe oxidants, even young cells can hemolyze.  Lab Finding Anemia is absent and peripheral blood finding are normal except during hemolytic attack or crises.  Bite cell  Heinz bodies (supravital stain)  Blister cells  Reticulocytosis  Increased bilirubin and lactate dehydrogenase enzyme (LDH- intracellular enzyme)  Decreased haptoglobin and hemoglobinuria  A lactate dehydrogenase (LDH or LD) is an enzyme found in nearly all living cells (animals, plants, and prokaryotes). LDH catalyzes the conversion of pyruvate to lactate and back, as it converts NADH to NAD+ and back. A dehydrogenaseis an enzyme that transfers a hydride from one molecule to another.  Bite Cells A degmacyte (aka "bite cell") is an abnormally shaped red blood cell with one or more semicircular portions removed from the cell margin. These "bites" result from the removal of denatured hemoglobin by macrophages in the spleen. Glucose-6-phosphate dehydrogenase deficiency, in which uncontrolled oxidative stress causes hemoglobin to denature and form Heinz bodies, is a common disorder that leads to the formation of bite cells. Bite cells can contain more than one "bite.“ The "bites" in degmacytes are smaller than the missing red blood cell fragments seen in schistocytes.[citation needed]  Blister cells Blister cells are the precursor of bite cells. In patients with G6PD deficiency, blister cells appear as red blood cells containing a peripherally located vacuole Blister Cells Heinz Bodies (supravital stain)  Note In affected individuals, with some enzyme deficiencies, enzyme activity is nearly normal during and a time after a hemolytic attack. 1) Hemolysis of old RBCs 2) The presence of reticulocytes (also transfusion of normal RBCs) Assay for G6PD should be performed 2 to 3 months after hemolytic episode (patient is not undergo hemolysis).  The timing of enzymatic testing and interpretation of the results is important for accurate diagnosis. With some enzyme deficiencies, enzyme activity is nearly normal in reticulocytes but decreases as the cell ages (e.g., G6PD@A- described in “G6PD Variants,” below). Thus, depending on the age distribution of the circulating cells and the degree of reticulocytosis, the enzyme content may appear normal. For this reason, the patient’s blood should not be collected for testing immediately after a hemolytic episode when most of the enzyme-deficient cells have been hemolyzed and there is a compensatory reticulocytosis. Diagnosis during or shortly after a hemolytic episode can be made if the blood is centrifuged and the older dense erythrocytes at the bottom of the column of blood are tested.2,3 If a patient has been recently transfused, testing should be delayed until the transfused cells are no longer present  Therapy In most cases of G6PD deficiency, patients are asymptomatic, and no treatment is indicated. In acute hemolytic episode, blood transfusion if needed and removal of the oxidizing agent (drug or fava bean) Prevention: avoid exposure to oxidizing agent and consumption of fava bean.  G6PD Case Report October 2017: 17 year old healthy young man took 30 grams (60 pills) of Optalgin (dipyrone) in an attempt to commit suicide. He was brought to Hospital around 12 hours later. Blood tests were “normal” (Hb 12, urea and creatinine normal, LDH not done). He told the ER doctors of G6PD deficiency. Psych consult discharged him. No medical follow up was scheduled.  X-inactivation (also called lyonization) is a process by which one of the copies of the X chromosome present in female mammals is inactivated. The inactive X chromosome is silenced by its being packaged in such a way that it has a transcriptionally inactive structure called heterochromatin. As nearly all female mammals have two X chromosomes, X- inactivation prevents them from having twice as many X chromosome gene products as males, who only possess a single copy of the X chromosome (see dosage compensation). The choice of which X chromosome will be inactivated is random in placental mammals such as humans, but once an X chromosome is inactivated it will remain inactive throughout the lifetime of the cell and its descendants in the organism. Unlike the random X- inactivation in placental mammals, inactivation in marsupials applies exclusively to the paternally derived X chromosome.  On the 6th day following ingestion, he felt bad, was pale and sleepy. G6PD Case Report He was brought to the ER at Hospital. Hb was 10, later fell to 6; Creatinine nearly 4 times normal, and LDH was nearly 10,000 (normal: 620). Diagnosis: acute hemolysis due to G6PD with acute renal failure Effective treatment was given (fluids, alkaline diuresis, blood transfusion, red cell exchange) Complete recovery  G6PD Case Report: Lessons Patient should have been referred for follow up visit in a day or two LDH measurement would have helped Drugs which are permissable in “therapeutic” doses can still be harmful if taken to excess by patients with G6PD. MOST IMPORTANT: Alertness by doctors! Hemolysis is often missed as diagnosis in hospitalized patients. Pyruvate Kinase (PK) Deficiency  Pyruvate Kinase (PK) Deficiency PK deficiency is the most common enzyme deficiency in the glycolytic pathway and the second most common erythrocyte enzyme deficiency (after G6PD). PK converts phosphoenol pyruvate to pyruvate with conversion of ADP to ATP. PK deficiency leads to cations imbalance and dehydration (echinocytes) and irregular contracted cells. The decreased deformability of echinocytes results in their sequestration in spleen.  Mechanism Because red blood cells cannot synthesize ATP, cellular death occurs. Cell death is by shrinking- this is called a 'dehydration at cellular level'. Due to the unavailability of adequate ATP, all active processes in the red blood cell comes to a halt. Sodium potassium ATPase pumps are the first to stop. Since the cell membrane is more permeable to potassium than sodium, potassium leaks out. Inter cellular fluid becomes hypertonic. Water moves down its concentration gradient out of the cells Finally cells shrink and die.  Lab findings in PK Deficiency Normocytic normochromic anemia Reticulocytosis Irregular contracted cells and echinocytes In contrast to G6PD deficiency, Heinz bodies and spherocytes are not found in PK deficiency. Bilirubin increased  Notes  Splenectomy can be the treatment  When performing enzyme test for PK, erythrocytes are separated from leukocytes because leukocytes contain more PK than erythrocytes. (erythrocytes PK gene located at chromosome 1 while leukocytes PK gene at chromosome 15) thus in PK deficiency the mutation in chromosome 1 only and normal PK activity in leukocytes). Echinocytes Immune Hemolytic Anemias When erythrocytes are destroyed prematurely by an immune mediated process (antibody and/or complement), the disorder is referred to as an immune hemolytic anemia (IHA). 1- Immune hemolytic anemias 1.1- Autoimmune hemolytic anemias Autoimmune hemolytic anemias (AIHAs) are caused by antibody production by the body against its own red cells. They are characterized by a positive direct antiglobulin test (DAT) also known as the Coombs' test and divided into 'warm‘ and 'cold' types according to whether the antibody reacts more strongly with red cells at 37°C or 4°C. LABORATORY IDENTIFICATION OF SENSITIZED RED CELLS When immune hemolytic anemia is suspected, tests to detect and identify the causative antibody are indicated. In general, two distinct techniques are used: 1. Agglutination in saline, which will detect antibodies of the IgM class 2. Antihuman globulin (AHG) test, which will detect antibodies of the IgG class and/or complement The complement system is made up of a large number of distinct plasma proteins that react with one another to opsonize pathogens and induce a series of inflammatory responses that help to fight infection The complement system is a part of the immune system that enhances (complements) the ability of antibodies and phagocytic cells to clear microbes and damaged cells from an organism, promotes inflammation, and attacks the pathogen's plasma membrane. It is part of the innate immune system, which is not adaptable and does not change over the course of an individual's lifetime.  1. Agglutination reactions  IgM antibodies can be detected by agglutination reactions between test sera and appropriate erythrocytes suspended in saline but IgG antibodies cannot  This difference in the ability of IgG and IgM to cause agglutination in saline can be explained on the basis of the difference in size of the two antibodies in relation to the zeta potential. The extracellular domain is heavily glycosylated and is responsible for most of the negative surface charge (zeta potential) that keeps red cells from sticking to each other and to the vessel wall The erythrocyte zeta potential is an electrostatic potential created by a difference in the charge density of the inner and outer layers of the ionic cloud around erythrocytes when they are suspended in saline. This force tends to keep the erythrocytes about 25 nm apart in solution. Thus, any antibody that causes agglutination of saline or plasma suspended cells must be large enough to span the 25 nm gap between cells. The IgM pentamer has a possible span of 35 nm; therefore, it can overcome the electrostatic forces separating the cells and cause agglutination. However, the maximum span of the IgG molecule is about 14 nm, and it cannot reach antigens on two separate cells to cause agglutination. Thus, detection of IgG antibodies requires a different technique using a substance that will connect the antibody molecules on separate cells and cause agglutination. The substance used is AHG, essentially an antiserum to human IgG. The AHG test, sometimes referred to as the Coombs test, is the specific laboratory procedure designed to detect erythrocytes sensitized with IgG and/or complement.  2. Antihuman globulin (AHG) test  The AHG test, sometimes referred to as the Coombs test, is the specific laboratory procedure designed to detect erythrocytes sensitized with IgG and/or complement.  AHG is a broad-spectrum antisera produced in rabbits that reacts against human immunoglobulin and complement.  It contains divalent antibodies that are capable of attaching to the Fc region of immunoglobulins or to complement components on two separate cells, thus bridging the distance between cells and leading to the lattice formation known as agglutination.  The two applications of the AHG test are: Direct: detects erythrocytes coated with antibody in vivo Indirect: detects antibodies in the plasma or serum The direct antiglobulin test (DAT, or direct Coombs test) detects erythrocytes that have been sensitized with antibody and/or complement in vivo AHG (anti-IgG and anticomplement) is added to the saline washed patient cells. Agglutination of patient cells by AHG is considered positive evidence for the presence of IgG and/or complement components on the cells due to in vivo coating. The indirect Coombs test is used in prenatal testing of pregnant women and in testing blood prior to a blood transfusion. It detects antibodies against RBCs that are present unbound in the patient's serum. In this case, serum is extracted from the blood sample taken from the patient. Then, the serum is incubated with RBCs of known antigencity; that is, RBCs with known reference values from other patient blood samples. If agglutination occurs, the indirect Coombs test is positive.  An indirect Coombs' test determines whether there are antibodies to the Rh factor in the mother’s blood The indirect antiglobulin test (IAT) is used to detect antibodies in the patient’s serum. A positive IAT indicates alloimmunization (immunization to antigens from another individual) and/or the presence of free autoantibody in the patient’s serum. In the IAT, free antibody is detected by incubating the patient’s serum with erythrocytes of known antigenic makeup The indirect antiglobulin test (IAT): Is used to detect antibodies in the patient’s serum. A positive IAT indicates alloimmunization and/or the presence of free autoantibody in the patient’s serum. Free antibody is detected by incubating the patient’s serum with erythrocytes of known antigenic makeup Only A and C All the above 1.1.1- Warm autoimmune hemolytic anemias The red cells are coated with immunoglobulin (Ig), usually immunoglobulin G (IgG) alone or with complement, and are therefore taken up by splenic macrophages which have receptors for the Ig Fc fragment. Therefore, prematurely destroyed, predominantly in the spleen.  Autoimmune hemolytic anemias are further classified as warm or cold hemolytic anemia based on clinical symptoms and on the optimal temperature at which the antibody reacts (in vivo and in vitro). Some antibodies react best at body temperature (37°C); the anemia they produce is termed warm autoimmune hemolytic anemia (WAIHA). About 70% of the AIHAs are of the warm type. The majority of warm autoantibodies are of the IgG class (most frequently IgG1) and cause extravascular hemolysis of the erythrocyte. A few warm-reacting autoantibodies of either the IgM or the IgA class have been identified. Warm autoimmune hemolytic anemias Clinical feature and laboratory diagnosis The disease may occur at any age, in either sex, and presents as a hemolytic anemia of varying severity. The spleen is often enlarged. It may occur in association with other diseases or arise in some patients as a result of some therapy.  The most common antibody involved in warm antibody AIHA is IgG, though sometimes IgA is found. The IgG antibodies attach to a red blood cell, leaving their FC portion exposed with maximal reactivity at 37 °C (versus cold antibody induced hemolytic anemia whose antibodies only bind red blood cells at low body temperatures, typically 28- 31 °C). The FC region is recognized and grabbed onto by FC receptors found on monocytes and macrophages in the spleen. These cells will pick off portions of the red cell membrane, almost as if they are taking a bite. The loss of membrane causes the red blood cells to become spherocytes. Spherocytes are not as flexible as normal RBCs and will be singled-out for destruction in the red pulp of the spleen as well as other portions of the reticuloendothelial system. The red blood cells trapped in the spleen cause the spleen to enlarge, leading to the splenomegaly often seen in these patients.  Therapy Corticosteroids. Cytotoxic drugs. High-dose intravenous immunoglobulin (IVIG). Plasma exchange and plasmapheresis. Splenectomy. Rituximab (monoclonal anti-CD20)  Corticosteroids. Initial therapy for patients with AIHA is often a course of immunosuppressive drugs such as corticosteroids, which are used to produce immunosuppression by decreasing lymphocyte proliferation and suppressing macrophage sequestration of sensitized cells by affecting the Fc receptors. Few patients undergo complete remission with this therapy, but most show a decrease in erythrocyte destruction. Response usually occurs within 10–14 days, and many patients require low-dose maintenance Cytotoxic drugs. A number of alternative therapies are available for patients who do not respond to corticosteroids and/or splenectomy.1,44 Cytotoxic drugs such as cyclophosphamide and azathioprine are used to cause general suppression of the immune system, which decreases synthesis of autoantibody. High-dose intravenous immunoglobulin (IVIG). This can block Fc receptors on macrophages and affect T and B cell function by increasing T suppressor cells or reducing B cell function. It has a variable success rate and is most often used as an adjunct therapy with corticosteroids or in selected patients with severe anemia who are refractory to drugs. Plasma exchange and plasmapheresis. This can dilute or temporarily remove the autoantibody from the patient’s circulation. It has been successful in reducing antibody load for a short time in some cases but is not a satisfactory long-term therapy.  Splenectomy. Indicated when severe anemia is unresponsive to medical therapies including glucocorticoid therapy or other cytotoxic drugs, splenectomy provides long-term remission for some patients. Removal of the spleen decreases the destruction of IgG-coated erythrocytes that would normally adhere to Fc receptors on splenic macrophages. If, however, the antibody concentration remains high, the destruction of sensitized erythrocytes may continue in the liver. Some evidence suggests that splenectomy can be more beneficial in patients with idiopathic WAIHA than in those with the secondary type. Rituximab has been used in the treatment of clonal B-cell malignancies, including chronic lymphocytic leukemia, in cases of autoimmune collagen vascular disease and in patients with cold agglutinin syndrome. It is recommended as a second-line therapy in patients with idiopathic and secondary WAIHA that are refractory to other treatments and is being used in place of splenectomy in many patients.18 Children appear to respond better than adults.40 Rituximab depletes the B cells that produce autoantibodies but does not apparently affect plasma cells or hematopoietic stem cells.  1.1.2- Cold autoimmune hemolytic anemias In these syndromes the autoantibody, attaches to red cells mainly in the peripheral circulation where the blood temperature is cooled, the antibody is usually IgM and binds to red cells best at 4°C. It is less clinically significant than warm. The secondary type associated with infectious disease is usually an acute, self-limiting form that has an onset 1–3 weeks after infection and resolves within 2–3 weeks. Cold hemolytic anemias, on the other hand, are usually due to the presence of an IgM antibody with an optimal thermal reactivity below 30°C. Hemolysis with cold-reacting antibodies results from the binding and activation of complement by IgM. The IgM antibody attaches to erythrocytes in the cold and fixes complement  Basic terms to remember related to antibodies Clinical significance: antibodies that are associated with decreased RBC survival Not clinically significant: antibodies that do not cause red cell destruction Cold reacting antibodies: agglutination best observed at or below room temp (optimal 4º C). Warm reacting antibodies: agglutination best observed at 37°C (body temperature)  1.2- Alloimmune hemolytic anemias In these anemias, antibody produced by one individual reacts with red cells of another. Two important situations are transfusion of ABO-incompatible (hemolytic transfusion reaction-HTR) blood and rhesus disease of the newborn (hemolytic disease of newborn-HDN)  Alloimmune hemolytic anemia occurs as a result of antibody development to an erythrocyte antigen that the individual lacks. When an individual is exposed to erythrocytes from another person, antigens on the transfused cells may be lacking on the erythrocytes of the recipient. Therefore, these antigens on the transfused cell can be recognized as foreign by the recipient’s lymphocytes and stimulate the production of antibodies (alloantibodies). In contrast to autoantibodies, these alloantibodies react only with the antigens on the transfused cells or cells from individuals who possess the antigen. The alloantibodies do not react with the individual’s erythrocytes.  Hemolytic transfusion reaction (HTR) A blood transfusion to patients can help them to restore strength and health. However, it is important that the blood is accurately matched to their blood type. If the blood type is not match, they can experience a transfusion reaction. HTR is a serious complication that can occur after a transfusion of blood. The red blood cells that were given in the transfusion are destroyed by the patient's immune system.  Hemolytic disease of newborn (HDN) Also known as hemolytic disease of the fetus and newborn (HDFN). It is an alloimmune condition that develops in a fetus, when the IgG molecules (one of the five main types of antibodies) produced by the mother pass through the placenta. These antibodies attack the red blood cells in the fetal circulation. 1.3- Drug-induce immune hemolytic anemias 1. Drug Adsorption (Hapten Type)  Some drugs such as penicillin are antigenic but lack the molecular size or complexity to initiate an immune response.  It is proposed that in the hapten/drug adsorption mechanism, the drug or its metabolites bind to proteins on the erythrocyte membrane, creating an immunogenic complex on the cell surface.  Antibodies are produced against the drug and react with the drug complex on the erythrocyte membrane. In each case, the hemolytic anemia gradually disappears when the drug is discontinued.  Only patients receiving high doses of intravenous penicillin develop significant penicillin coating on their cells, and only a small percent of these individuals will have a positive DAT; even fewer develop hemolysis.  The hemolytic anemia usually develops over a 7- to l0-day period.  Hemolysis is extravascular and mediated by Fc receptors on splenic macrophages.  Spherocytes can be present, and the reticulocyte count is usually elevated. 2. Immune Complex Formation  In this mechanism, a drug or drug metabolite with a low affinity for the cell membrane combines with a plasma protein, forming a new antigenic complex in the plasma and stimulating either IgM or IgG antibodies.  A drug/antidrug immune complex forms in the plasma, and this immune complex attaches to the erythrocyte in a nonspecific (nonimmune) manner (usually via the Fc portion of the antibody).  The attached immune complex usually has the ability to activate complement Quinidine is a pharmaceutical agent that acts as a class I antiarrhythmic agent (Ia) in the heart 3. Autoantibody Induction  In approximately 10–20% of patients receiving the antihypertensive drug Aldomet a positive DAT develops after about 3–6 months.  However, hemolytic anemia develops in only 1% of these patients.  The mechanism by which antibody production is induced is unknown; however, evidence indicates that the drug alters normal erythrocyte antigens, so they are no longer recognized as self. Methyldopa, sold under the brand name Aldomet among others, is a medication used for high blood pressure. 4. Membrane Modification  In addition to causing a positive DAT and immune hemolysis due to the drug adsorption mechanism, cephalosporins, especially the first-generation drugs, are capable of modifying the erythrocyte membrane so that normal plasma proteins including immunoglobulins and complement bind to the membrane in a nonimmunologic manner.  The adsorbed antibodies are not specific to any drug or drug/cell complex. The DAT is positive with polyspecific antisera and can be positive or negative with anti-IgG and anti-C3.  Quinidine is a pharmaceutical agent that acts as a class I antiarrhythmic agent (Ia) in the heart Methyldopa, sold under the brand name Aldomet among others, is a medication used for high blood pressure. Autoantibody Induction In approximately 10–20% of patients receiving the antihypertensive drug Aldomet a positive DAT develops after about 3–6 months. However, hemolytic anemia develops in only 1% of these patients. The mechanism by which antibody production is induced is unknown;  Acquired Hemolytic Anemias 2- Red cell fragmentation syndromes These arise through physical damage to red cells either on abnormal surfaces (e.g. artificial heart valves) or arteriovenous malformations as a microangiopathic hemolytic anemia. This is caused by red cells passing through abnormal small vessels.  Acquired Hemolytic Anemias 3- Infection Infections can cause hemolysis in a variety of ways. They may precipitate an acute hemolytic crisis in G6PD deficiency. Malaria causes hemolysis by extravascular destruction of parasitized red cells. But sometimes may cause direct intravascular lysis.  Acquired Hemolytic Anemias 4- Chemical agent 5- Secondary hemolytic anemias Liver disease (acanthocytes/spur cell anemia) 6-Paroxysmal nocturnal hemoglobinuria (PNH)  Paroxysmal nocturnal hemoglobinuria (PNH) PNH is a rare, acquired, clonal disorder of marrow stem cells. PNH is the only hemolytic anemia caused by an acquired (rather than inherited) intrinsic defect. Somatic mutation at level of the multipotential stem cell; abnormalities in WBC, PLTs and RBCs. In PNH, there is a deficiency in the synthesis of the glycosylphosphatidylinositol (GPI) anchor, in which carbohydrate- containing linker is glycosidically bound to the inositol residue.  Paroxysmal nocturnal hemoglobinuria (PNH) Type I Little or no hemolysis by complement; nearly ormal GPI-linked protein expression Type II Moderately sensitive to complement lysis; intermediate GPI-linked protein expression Type III Highly sensitive to complement lysis; no expression of GPI-linked proteins  Paroxysmal nocturnal hemoglobinuria (PNH) is a rare acquired disorder of the erythrocyte membrane. The membrane defect makes the cell abnormally sensitive to lysis by complement. The disease derives its name from the classic pattern of intermittent bouts of intravascular hemolysis and nocturnal hemoglobinuria. The condition is exacerbated during sleep and remits during the day. However, many patients have chronic hemolysis that is not associated with sleep and with no obvious hemoglobinuria. This structure is required to attach several surface proteins to the cell membrane. These proteins are used to protect the cell from destruction by the complement system, and without these anchors, the cells are more easily targeted by the complement system. Render red cells sensitive to lysis by complement and the result is chronic intravascular hemolysis. Hemosiderinuria is a constant feature and can give rise to iron deficiency which may increase the anemia. Paroxysmal nocturnal hemoglobinuria (PNH), sometimes referred to as Marchiafava- Micheli syndrome, is a rare, acquired, potentially life-threatening disease of the blood characterized by complement-induced intravascular hemolytic anemia. Any of the causes of hypersplenism (increased activity of the spleen), such as portal hypertension. Acquired hemolytic anemia is also encountered in burns and as a result of certain infections. Lead poisoning resulting from the environment causes non-immune hemolytic anemia. Runners can suffer hemolytic anemia due to "footstrike hemolysis", owing to the destruction of red blood cells in feet at foot impact. Low-grade hemolytic anemia occurs in 70% of prosthetic heart valve recipients, and severe hemolytic anemia occurs in 3%.  Treatment of PNH Transfusion therapy may be needed (treats anemia, and suppresses the production of PNH cells by the bone marrow) The drug eculizumab was approved for the treatment of PNH. It is a monoclonal antibody that protects blood cells against immune destruction by binding to a specific complement component and preventing the natural immune response. This drug binds to C5 and preventing its activating conversion to C5a, thus preventing formation of the membrane attack complex. a humanized monoclonal antibody that is a terminal complement inhibitor  Explain why MCV, MCH, and MCHC may be falsely increased when blood from someone with Cold hemolytic anemia is tested using an automated counter ?? If the blood is cool, the IgM antibodies cause agglutination of the cells. As these clumps of cells are measured for MCV, they are measured as a single cell and thus appear macrocytic. Hematocrit (which is calculated) is incorrect because of the incorrect erythrocyte count. Hemoglobin is correct because this parameter is determined by lysis of the cells. The erythrocyte count is falsely decreased because the clumps are counted as one cell. MCH, which is derived from the hemoglobin and erythrocyte count, then is falsely elevated as is the MCHC, which is derived from the hemoglobin and hematocrit values.  Diagnostic flowchart for hemolytic diseases AIHA: autoimmune hemolytic anemia; DHTR: delayed hemolytic transfusion reactions; CDA: congenital dyserythropoietic anemia; PNH: paroxysmal nocturnal hemoglobinuria. giant multinucleated erythroblasts; CDAIV, multinucleate erythroblasts; and CAD deficiency, binucleate CDAII-like precursors. Megaloblastic Anemia  Megaloblastic Anemia Macrocytic anemia characterized by large erythrocytes (MCV > 100fL) with an increased MCH and a normal MCHC. The megaloblastic anemia is due to abnormal DNA synthesis (nuclear maturation defect >>>>> Delay nuclear maturation). Most often due to vitamin B12 or folate deficiency. Megaloblastic anemia is typically a macrocytic, normochromic anemia.  Megaloblastic anemia (or megaloblastic anaemia) is an anemia (of macrocytic classification) that results from inhibition of DNA synthesis during red blood cell production. When DNA synthesis is impaired, the cell cycle cannot progress from the G2 growth stage to the mitosis (M) stage. This leads to continuing cell growth without division, which presents as macrocytosis Megaloblastic anemia is typically a macrocytic, normochromic anemia. The MCV is usually higher than 100 fL and can reach a volume of 140 fL. However, an increased MCV is not specific for megaloblastic anemia. The MCH is increased because of the large cell volume, but the MCHC is normal. In vitamin b12 deficiency, a macrocytosis can precede the development of anemia by months to years. On the other hand, the MCV can remain in the reference range. Epithelial changes in the gastrointestinal tract can cause iron absorption to be impaired. If an iron deficiency—which characteristically produces a microcytic, hypochromic anemia—coexists with megaloblastic anemia, macrocytosis can be masked and the MCV can be in the normal range.9 Other conditions that have been shown to coexist with megaloblastic anemia in the absence of an increased MCV include thalassemia, chronic renal insufficiency, and chronic inflammation or infection.8 MCH= Hb/RBC count MCHC=Hb/Hct (Hct= RBC count*MCV)=MCH/MCV in megaloblastic anemia MCV is high so, MCHC is normal/low Ultimately hemoglobin increases relative to the size of the cell so that the mean corpuscular hemoglobin concentration (MCHC) stays within the normal range and megaloblastic anemias are normochromic  Artifacts causing macrocytosis Macrocytosis detected by automated cell counters is not always apparent microscopically on stained blood smears. In some cases, the erythrocyte size on automated counters is falsely elevated due to hyperglycemia, cold agglutinins, and extreme leukocytosis. These causes of false macrocytosis need to be differentiated from true macrocytosis. The most common cause of true macrocytosis is alcoholism. Other causes include folate and cobalamin deficiencies, drugs including chemotherapy, reticulocytosis due to hemolysis or bleeding, myelodysplasia, liver disease, and hypothyroidism.  Megaloblastic Anemia Anemia is due to ineffective erythropoiesis resulting from disrupted DNA synthesis. The anemia is called megaloblastic in an attempt to describe the giant appearing erythroid precursors (megaloblast) in the bone marrow. B12 deficiency could be the result of a deficiency in intrinsic factor (IF),which secreted from gastric parietal cells, or due to nutritional deficiency. Folic acid deficiency, on the other hand, is most often due to an inadequate dietary intake.  Parietal cells (also known as oxyntic or delomorphous cells), are the epithelial cells that secrete hydrochloric acid(HCl) and intrinsic factor Intrinsic factor (IF), also known as gastric intrinsic factor (GIF), is a glycoprotein produced by the parietal cells of the stomach. It is necessary for the absorption of vitamin B12 (cobalamin) later on in the small intestine. In humans, the gastric intrinsic factor protein is encoded by the GIF gene.  B12 is required by enzymes for two reactions: the conversion of methylmalonyl CoA to succinyl CoA, and the conversion of homocysteine to methionine. In the latter reaction, the methyl group of 5-methyltetrahydrofolate is transferred to homocysteine to produce tetrahydrofolate and methionine. This reaction is catalyzed by the enzyme methionine synthase with B12 as an essential cofactor. During B12deficiency, this reaction cannot proceed, which leads to the accumulation of 5-methyltetrahydrofolate. This accumulation depletes the other types of folate required for purine and thymidylate synthesis, which are required for the synthesis of DNA. Inhibition of DNA replication in red blood cells results in the formation of large, fragile megaloblastic erythrocytes. The neurological aspects of the disease are thought to arise from the accumulation of methylmalonyl CoA due to the requirement of B12 as a cofactor to the enzyme methylmalonyl CoA mutase. Cobalamin is an essential cofactor for two enzymes in human cells: methionine synthase, which catalyzes the conversion of homocysteine to methionine, and methylmalonyl-CoA synthase, which catalyzes the conversion of methylmalonyl CoA to succinyl CoA Vitamin B12, or cobalamin, is an essential vitamin for the proper functioning and development of the brain and the nerve cells. It plays an important role in the maintenance of the sheaths that cover and protect the nerves of the central and the peripheral nervous system, ensuring proper and faster nerve-impulse transmission. A fatty substance called myelin is essential for the formation of these sheaths. Vitamin B12 plays a significant role in the synthesis and maintenance of myelin. The neurological problems caused by vitamin B12 deficiency later in life are due to the damage caused to the myelin sheath. (1) Dietary vitamin B12 is normally bound to proteins in food and is provided by food products of animal origin including milk, eggs, and meat. (2) Pepsin and acid pH in the stomach will degrade these food proteins and release vitamin B12. (3) The vitamin B12 that is now free then binds to one of the three vitamin B12 binding proteins, called haptocorrin, which is produced by the salivary glands and the parietal cells in the stomach. HC is the preferred binding protein for cobalamin released from food. The HC–cobalamin complex protects the cobalamin from degradation by the hydrochloric acid in the stomach. In the duodenum, pancreatic proteases degrade the haptocorrin releasing cobalamin. The released cobalamin quickly binds to intrinsic factor (IF), which resists pancreatic degradation. (4) The IF–cobalamin complex resists digestion and passes through the jejunum into the ileum where it binds the specific IF receptor (cubulin) on the microvilli of ileal mucosal cells. Binding requires a pH of 5.4 or higher as well as calcium. Following attachment of the IF–cobalamin complex to cubulin, the entire complex is taken into the mucosal cell by endocytosis. (5) Vitamin B12 then enters the blood while IF is degraded and is bound to another binding protein, transcobalamin: Transcobalamin (TC) is produced in many types of cells, including hepatocytes, enterocytes, macrophages, and hematopoietic precursors in the bone marrow. Although TC carries only a small fraction 20-30%of the total cobalamin in the plasma; The complex is known as holotranscobalamin (Active B12) while the majority of vitamin B12 (70-80%) in blood is bound to haptocorrin. (6) Holotranscobalamin (Active B12) is the biologically active fraction of vitamin B12 in the blood as it is in only this form that vitamin B12 is delivered to all the cells of the body. (7) Vitamin B12 absorbed in the intestine subsequently gets transported to the liver via the portal system. (8) There is extensive enterohepatic circulation of vitamin B12 and B12 is transported from the liver, via the bile duct, to the duodenum. Any issue in any step along the way could lead to problems. Obviously, you can see how a diet lacking in sufficient animal products would lead to a higher risk of vitamin B12 deficiency. However, a dietary cause is a relatively uncommon reason. More common is malabsorption due to a number of gastrointestinal risk factors: Atrophic gastritis, which increases with age, impairs both the production of the acid and enzymes needed to break down food, and the production of intrinsic factor. Pancreatic insufficiency, and of course any surgery which removed any part of the stomach or ileum, could all lead to malabsorption. Intestinal diseases such as Crohn’s and celiac disease could cause problems. Long-term use of acid suppressants, like proton pump inhibitors and H2 antagonists. These are some of the most widely prescribed drugs among the elderly. In true pernicious anemia, where there’s an autoimmune component, there are three different types of antibodies. Those which bind to the B12-IF complex, preventing uptake, those which bind to IF itself, preventing the binding with B12, and those which bind to parietal cells, preventing the production of IF to begin with. In PA patients, antibodies form and attack either the intrinsic factor (IF), or the gastric parietal cells, which produce IF in first place. Thus, the two types of pernicious anemia antibodies are the gastric parietal cell antibodies (GPCAs, or just PCAs) and the intrinsic factor antibodies (IFAs). There are two types of IFAs: Type I: Blocking antibodies. These inhibit B12 from binding to IF, blocking the formation of the B12/IF complex that is meant to carry the B12 to the small intestine. Type II: Binding antibodies. These bind to the B12/IF complex and prevent its absorption. They exist almost only in those who already have type I antibodies, which is why doctors will normally test just for type I.  Clinical Findings Neurological disturbances occur only in vitamin B12 deficiency. Neurological symptoms has been reported to occur even before anemia and macrocytosis. Tingling and numbness in the extremes.  Cobalamin is an essential cofactor for two enzymes in human cells: methionine synthase, which catalyzes the conversion of homocysteine to methionine, and methylmalonyl-CoA synthase, which catalyzes the conversion of methylmalonyl CoA to succinyl CoA Vitamin B12, or cobalamin, is an essential vitamin for the proper functioning and development of the brain and the nerve cells. It plays an important role in the maintenance of the sheaths that cover and protect the nerves of the central and the peripheral nervous system, ensuring proper and faster nerve-impulse transmission. A fatty substance called myelin is essential for the formation of these sheaths. Vitamin B12 plays a significant role in the synthesis and maintenance of myelin. The neurological problems caused by vitamin B12 deficiency later in life are due to the damage caused to the myelin sheath.  Peripheral Blood  Macrocytic normochromic anemia.  MCV increased, MCH increase and MCHC is normal.  Because the abnormality is a nuclear maturation defect, the megaloblastic anemia affect all three blood lineages (RBCs, WBCs & Platelets). This is unlike other anemias that typically involve only erythrocytes.  MCH= Hb/RBC count MCHC=Hb/Hct (Hct= RBC count*MCV)=MCH/MCV in megaloblastic anemia MCV is high so, MCHC is normal/low Cont… The distinguishing features of megaloblastic anemia in blood film are: Oval macrocytes (macroovalocyte) Howell- Jolly bodies Hyper-segmented neutrophil (> 5 nuclear lobes) Erythrocytes that contain Cabot ring can be seen.  Hyper segmented neutrophils are larger than normal neutrophils Megaloblastic Anemia  Megaloblastic anemia A) Cabot ring (they are believed to be remnants from a mitotic spindle) B) Howell-Jolly body  Bone Marrow The bone marrow with megaloblastic erythroid precursors. Because the cytoplasmic maturation continues in normal fashion, the normoblasts contain more cytoplasm with more mature appearance relative to the maturity of the nucleus.  Hypersegmented PMN Megaloblastic anemia Megaloblastic BM  Schilling test The Schilling test is used to determine whether the body absorbs vitamin B12 normally or not. If not, whether the patient has pernicious anemia or other problems with B12 absorption. As well as, whether the problem is due to nutritional deficiency.  Pernicious anemia (also known as Biermer's anemia, Addison's anemia, or Addison– Biermer anemia) is one of many types of the larger family of megaloblastic anemias. One way pernicious anemia can develop is by loss of gastricparietal cells, which are responsible, in part, for the secretion of intrinsic factor, a protein essential for subsequent absorption of vitamin B12 in the ileum.  Schilling Test Stage 1: Oral radiolabeled vitamin B12 plus intramuscular unlabeled vitamin B12 IM injection is done to saturate all vitamin B12 receptors in the body. Therefore, if the oral radiolabeled vitamin B12 is absorbed in the intestine, it will be prevented from binding to their receptor in the tissues (especially in the liver). Accordingly, it will pass into the urine. If the radiolabeled B12 is detected in the urine So, there is normal absorption of B12 and the problem is loss of B12 intake (nutritional deficiency).  in the first part of the test, the patient is given radiolabeled vitamin B12 to drink or eat. The most commonly used radiolabels are 57Co and 58Co. An intramuscular injection of unlabeled vitamin B12 is given an hour later. This is not enough to replete or saturate body stores of B12 that requires about 10 B12 injections over some length of time. The purpose of the single injection is to temporarily saturate B12 receptors in the liver with enough normal vitamin B12 to prevent radioactive vitamin B12binding in body tissues (especially in the liver), so that if absorbed from the G.I. tract, it will pass into the urine. The patient's urine is then collected over the next 24 hours to assess the absorption. Normally, the ingested radiolabeled vitamin B12 will be absorbed into the body. Since the body already has liver receptors for transcobalamin/vitamin B12 saturated by the injection, much of the ingested vitamin B12 will be excreted in the urine. A normal result shows at least 10% of the radiolabeled vitamin B12 in the urine over the first 24 hours. In patients with pernicious anemia or with deficiency due to impaired absorption, less than 10% of the radiolabeled vitamin B12 is detected. The normal test will result in a higher amount of the radiolabeled cobalamin in the urine because it would have been absorbed by the intestinal epithelium, but passed into the urine because all hepatic B12 receptors were occupied. An abnormal result is caused by less of the labeled cobalamin to appear in the urine because it will remain in the intestine and be passed into the feces.  Normal ranges A normal result shows at least 10% of the radiolabeled vitamin B12 in the urine over the first 24 hours. In patients with pernicious anemia or with deficiency due to impaired absorption, less than 10% of the radiolabeled vitamin B12 is detected. The normal test will result in a higher amount of the radiolabeled cobalamin in the urine because it would have been absorbed by the intestinal epithelium but passed into the urine because all hepatic B12 receptors were occupied. An abnormal result is caused by less of the labeled cobalamin to appear in the urine because it will remain in the intestine and be passed into the feces.  Stage 2: vitamin B12 and intrinsic factor If an abnormality is found, i.e. the B12 in the urine is only present in low levels, the test is repeated, this time with additional oral intrinsic factor. If this second urine collection is normal, this shows a lack of intrinsic factor production, or pernicious anemia. A low result on the second test implies abnormal intestinal absorption (malabsorption), If the radiolabeled B12 is not detected in urine, there is malabsorption Stage II: Oral radiolabeled vitamin B12 and intrinsic factor (with unlabeled IM B12) If the radiolabeled B12 is detected in the urine So, the problem is in the secretion of IF from parietal cells Pernicious anemia  Pernicious anemia is only one specific cause of vitamin malabsorption, which also can be caused by a loss of IF secondary to gastrectomy or secondary to diseases that prevent binding of the IF-B12 complex in the ileum. An iron deficiency usually precedes vitamin deficiency in patients who have had a gastrectomy. Diseases that can affect the absorption of the IF- B12 complex in the ileum include Crohn’s disease, tropical sprue, celiac disease, and surgical resection of the ileum. stage II: You are given radioactive B12 along with intrinsic factor. Intrinsic factor is a protein produced by cells in the stomach lining. The body needs it so the intestines can absorb vitamin B12. Stage II of the test can tell whether a low vitamin B12 level is caused by problems in the stomach, preventing it from producing intrinsic factor.  If the radiolabeled B12 is not detected in urine, there is other problem in B12 absorption Stage 3: Oral radiolabeled vitamin B12 and antibiotics Is used for detection of patients with: bacterial overgrowth syndrome Stage III: This test is done after you have taken antibiotics for 2 weeks. It can tell whether abnormal bacterial growth has caused the low vitamin B12 levels. Exocrine pancreatic insufficiency (EPI) is the inability to properly digest food due to a lack of digestive enzymes made by the pancreas. EPI is found in humans afflicted with cystic fibrosis Stage 4: Oral radiolabeled vitamin B12 and pancreatic enzymes Is used for detection of patients with pancreatitis. Schilling Test  First, the test results are not valid with the presence of renal disease. The patient could have been able to absorb the vitamin but cannot filter the excess vitamin efficiently because of abnormal kidney function. Second, incomplete collection of urine invalidates the results. Incontinence or inability to empty the bladder gives false low values even when absorption was normal. Spuriously low urinary excretion in part II can also be due to the inability to absorb IF-B12 because of megaloblastoid epithelial changes in the gut. The Schilling test is seldom used now in this country because of the difficulties in using radioisotopes and the inconvenience of the test for the patient. An additional problem is that the absorption of crystalline vitamin b12 used in the oral dose can differ from the absorption of vitamin in food  The hemoglobin and erythrocyte count range from normal to very low. The erythrocyte count is occasionally less than 1.0 * 106 /mcL. However, anemia is not always evident. In one study of 100 patients with confirmed cobalamin deficiency, only 29% had a hemoglobin of less than 12 g/dL.11 This is significant because neurologic symptoms can be present even if the MCV and/or hematocrit are normal.12 Because the abnormality is a nuclear maturation defect, the megaloblastic anemias affect all three blood cell lineages: erythrocytes, leukocytes, and platelets. One study showed that in patients with renal disease, iron deficiency, or chronic disease with a normal or decreased MCV and 1% hypersegmented neutrophils, 94% had vitamin B12 or folic acid deficiency urinary excretion of formiminoglutamic acid (FIGLU)- biomarker for intracellular levels of folate. The FIGLU test is used to identify vitamin B₁₂ deficiency, folate deficiency, and liver failure or liver disease. Folic acid deficiency results in inability to degrade a formiminoglutamic acid (FIGLU) to glutamic acid so that FIGLU accumulates in excessive amounts and is excreted in the urine.  Teardrop cells (dacrocytes) are frequently associated with infiltration of the bone marrow by fibrosis, granulomatous inflammation, or hematopoietic or metastatic neoplasms. They can also be seen in patients with splenic abnormalities, vitamin B12 deficiency, and some other forms of anemia. A Howell–Jolly body is a cytopathological finding of basophilic nuclear remnants (clusters of DNA) in circulating erythrocytes Algorithm of diagnosis of vitamin B12 and folic acid deficiency using laboratory tests.  Pernicious anemia is often associated with autoimmune disease. Up to 90% of PA patients have antibodies against parietal cells. However, these antibodies are not specific for pernicious anemia and are also found in patients with gastritis, thyroid disease, and Addison’s disease. On the other hand, serum antibodies against intrinsic factor are found in about 75% of PA patients and are highly specific for PA. Antibodies directed against the patient’s own cells or proteins are found. Autoantibodies found in pernicious anemia (PA) are of two types, blocking and binding. Blocking antibodies prevent the formation of the intrinsic complex, and binding antibodies prevent attachment and absorption of into the ileum mucosal epithelial cell. Antibodies against intrinsic factor are detected in up to 75% of PA patients. The majority of PA patients demonstrate antibodies against the parietal cells in the floor of the stomach. Patients with PA can have one or both types of antibodies.  Elevated blood Hcy represents folate and Cbl deficiency, whereas MMA is the sole determinant of Cbl function [, , ]. Cbl plays a catalytic role by supplying methyl groups in the methionine cycle for protein and DNA synthesis, whereas folate participates in cell proliferation via purine, thymidylate and various co-dependent pathways Algorithm that can be used to determine the cause of megaloblastic anemia using laboratory tests. Analysis can begin with a serum cobalamin assay. If the results are in the low normal range (150–400 pg/mL) or the patient has unexplained neurologic symptoms, measurement of cobalamin metabolic intermediates, methylmalonic acid (MMA), and homocysteine is suggested. If both are elevated cobalamin deficiency is considered. If MMA is normal and homocysteine is elevated, folate deficiency is probable. RBC or serum folate also can be measured, especially if cobalamin is within the reference interval. If these test results conflict with the clinical diagnosis, therapeutic trials with cobalamin can be used. Bone marrow is rarely performed for diagnostic purposes but can be necessary in some cases. The bone marrow shows megaloblastic features in both folate and cobalamin deficiencies.  A specific test that measures the increased excretion of methylmalonic acid (MMA) in the urine indirectly indicates decreases in vitamin concentration. Homocysteine increases in the plasma of patients with vitamin or folate deficiency. Monitoring serum levels can serve as an early detector of vitamin deficiency. Many recent studies have concluded that MMA and homocysteine are the most sensitive and specific indicators of vitamin deficiency Requirements: About 3–5 mcg of cobalamin per day is needed to maintain normal biochemical functions. It is estimated that only about 70% of cobalamin intake is absorbed, which suggests that the diet should include 5–7 mcg of the vitamin per day. This amount is available in a regular “balanced” diet but not in a strict vegetarian diet. Cobalamin stores (about 5,000 mcg) are sufficient to provide the normal daily requirement for about 1,000 days. Therefore, it takes several years to develop a deficiency if no cobalamin is absorbed from the diet. About half of the vitamin is stored in the liver, and the rest is located in the heart and kidneys.1 Elevated Hcy-MMA levels on pediatrics health have been associated with irreversible neurological deterioration, neural tube defects (NTD), anaemia, renal dysfunction, hyperhomocysteinemia, methylmalonic acidemia and long- term adverse effects such as impaired cognitive function and mental behaviour, inability to bear stressful events with the commencement of suicidal tendencies which is rising worldwide. Aplastic anemia and bone marrow failure WHAT IS APLASTIC ANEMIA ? Aplastic anemia (AA) is a hematologic disorder characterized by pancytopenia on peripheral blood smear and a markedly hypocellular or acellular marrow (aplasia). ( i.e It’s an anemia due to failure of the bone marrow to produce red and white blood cells as well as platelets.) It is classified into primary (congenital or acquired) or secondary types. Aplastic anemia occurs in all age groups, with two peaks one In childhood and the other is found in the 20 to 25-year-old age group. The male-to-female ratio is approximately 1:1. incomplete, retarded, or defective development, or cessation of the usual regenerative process The underlying defect in all causes appears to be a reduction in the number of hemopoietic stem cells, and a fault in the remaining stem cells, or an immune reaction against them, which makes them unable to divide and differentiate properly to populate the bone marrow Causes of aplastic anemia  Fanconi anemia is an inherited disease caused by mutations in certain genes, known as FA genes. Inherited These genes provide instructions to help the body repair certain types of DNA damage. The cells of healthy people aplastic often repair DNA damage, but cells affected by Fanconi anemia anemia cannot make these repairs.  In people who have Fanconi anemia, certain cells may die or stop working properly. Fanconi Fanconi anemia can lead to serious complications anemia such as bone marrow failure, and cancers such as acute myeloid leukemia (AML) is one of the complications. To diagnose Fanconi anemia, you may look for dark spots on the skin called café au lait spots.  Café-au-lait spots are light to dark brown pigmented birthmarks that commonly appear on a newborn's skin. Spots can change in size and number over time. More than six café-au-lait spots can be a sign of an underlying genetic condition like neurofibromatosis type 1 (NF1). mutation of neurofibromin, a gene on chromosome 17 that is responsible for production of a protein which is needed for normal function in many human cell types. NF-1 causes tumors along the nervous system which can grow anywhere on the body.  Fanconi Anemia (FA) FA is the result of a genetic defect in a cluster of proteins responsible for DNA repair. As a result, the majority of FA patients develop cancer, and develop bone marrow failure (the inability to produce blood cells). 2/3 of FA patients have abnormalities of the skin, arms, kidneys and developmental disabilities.   Dyskeratosis congenita is a rare genetic form of bone Dyskeratosis marrow failure, the inability congenita of the marrow to produce sufficient blood cells. Inherited  Patients presented with the aplastic anemia triad of: 1. Abnormal skin 2. Malformation (dystrophy) of the nails 3. White, thickened patches on the mucous membranes of the mouth -(oral leukoplakia)  Dyskeratosis is Latin and means the irreversible degeneration of skin tissue, and congenita means inborn. Oral leukoplakia is a white patch or plaque that develops in the oral cavity and is strongly associated with smoking. Patients have very short germline telomeres, and approximately half have mutations in one of six genes encoding proteins that maintain telomere function. Accurate diagnosis of DC is critical to ensure proper clinical management because patients with DC and bone marrow failure do not respond to immunosuppressive therapy and may have increased morbidity and mortality associated with hematopoietic stem cell transplantation. Amegakaryocytic  Congenital thrombocytopenia amegakaryocytic thrombocytopenia (CAMT) is a rare disease. Inherited aplastic anemia  Symptoms for congenital amegakaryocytic thrombocytopenia include bruising and bleeding  Acquired amegakaryocytic thrombocytopenia is a rare blood disorder that causes severe thrombocytopenia with no other blood abnormalities. It is so named because the level of large bone marrow cells that produce platelets , called megakaryocytes, are significantly lower or absent. Acquired amegakaryocytic thrombocytopenia previously diagnosed as idiopathic thrombocytopenic purpura in a patient with hepatitis C virus infection Type 1 results from a stop codon or frameshift mutation, causing a loss of the intracellular domain of the thrombopoietin receptor with complete loss of function of the receptor. This results in early progression to bone marrow failure, with the mean age being one year and 11 months. Type 2 results from a splicing defect or amino acid substitution, which can affect the MPL receptor's glycosylation and result in an inability to react with thrombopoietin (THPO). These mutations can also cause a loss of hydrogen bonds within the MPL receptor, making it unstable.   is characterized by an autosomal recessive mode of inheritance.  The gene that is mutated in this Inherited syndrome: aplastic  SBDS (ribosome maturation protein)- anemia This gene encodes a highly conserved protein that plays an essential role in ribosome biogenesis. Shwachman-  The main characteristics of the diamond syndrome are exocrine pancreatic syndrome dysfunction, hematologic abnormalities and growth retardation.  Shwachman-Diamond syndrome (SDS) affects many parts of the body, particularly the bone marrow, pancreas, and skeletal system. Symptoms include the inability to digest food due to missing digestive enzymes, low muscle tone, and anemia. Other symptoms include skeletal findings and intellectual disability. Children with SDS may have feeding difficulties, slow growth, and frequent infections. People with SDS are at increased risk for blood cancers.  Acquired aplastic anemia  The acquired causes which consist about 80% of causes of aplastic anemia.  Aplastic anemia here can be caused by exposure to certain chemicals, drugs, radiation, infection, immune disease; in about half the cases, a definitive cause is unknown.  Idiopathic (Acquired)  Accounting most of acquired cases.  In most cases, the hematopoietic tissue is the target of an immune process.  Pathophysiology of aplastic anemia  AA is thought to be mediated by abnormally activated T lymphocytes and their secreted lymphokines, which subsequently result in HSC dysfunction and BM destruction.  Over-production of pro-inflammatory cytokines, including IFN-γ, TNF-α and other regulators, inhibits the hematopoietic system hematopoietic progenitor and and leads to cell apoptosis through the stem cells Fas/FasL signaling pathway.  In addition, IFN-γ could induce PD-L1 expression on T cells, NK cells and dendritic cells, which then binds to PD-1 to induce apoptosis and reduce immune tolerance.  PD-L1 is an immune inhibitory receptor ligand that leads to T cell dysfunction and apoptosis by binding to its receptor PD-1, which works in braking inflammatory response and conspiring tumor immune evasion. Programmed death cell receptor 1 (PD-1) is expressed on T cells upon T cell receptor (TCR) stimulation. PD-1 ligand 1 (PD-L1) is expressed in most tumor environments, and its binding to PD-1 on T cells drives them to apoptosis or into a regulatory phenotype. PD-1 is a cell surface receptor on T cells and B cells that has a role in regulating the immune system's response to the cells of the human body by down-regulating the immune system and promoting self-tolerance by suppressing T cell inflammatory activity. Usually, AA is a result of HSPC destruction targeted by autoreactive cytotoxic T cells. Tissue expression of PD-L1 mediates peripheral T cell tolerance Programmed death 1 (PD-1), an inhibitory receptor expressed on activated lymphocytes, regulates tolerance and autoimmunity. Immune tolerance, or immunological tolerance, or immunotolerance, is a state of unresponsiveness of the immune system to substances or tissue that have the capacity to elicit an immune response in a given organism. SIGNS AND SYMPTOMS Fatigue Shortness of breath Rapid or irregular heart rate Pale skin Frequent or prolonged infections Unexplained or easy bruising Nosebleeds and bleeding gums Prolonged bleeding from cuts Skin rash Dizziness Headache Fever  Secondary Radiation Cytotoxic drugs Chemicals (benzene) Some viral infections.  Immunosuppressive therapy presumably eliminates an abnormal population of activated T lymphocytes that produce tumor necrosis factor (TNF), substances known to inhibit hematopoiesis.  One mechanism of inhibition is likely cell killing through induction of apoptosis. Both and TNF induce overexpression of Fas (a cell membrane receptor) by progenitor cells. When the Fas receptor is activated by binding with its ligand, apoptosis is initiated.  In Epstein-Barr viral infections as well as other viral infections, the virus can infect the hematopoietic stem cell, triggering an immune response. Cytotoxic lymphocytes then destroy the stem cell. Other mechanisms of stem cell damage in viral infections have been postulated, including direct cytotoxicity of the virus and inhibition of cellular proliferation and differentiation.  Clinical Feature Symptoms are related to anemia, neutropenia or thrombocytopenia. Infections are common and generalized infections are frequently life-threatening.  Bleeding gums, epistaxis and menorrhagia are the most frequent hemorrhagic manifestations With symptoms of anemia.  Laboratory Findings Anemia (normochromic normocytic). The reticulocyte count is usually low in relation to the degree of anemia. Leukopenia (usually below 1.5 x 109/L). Thrombocytopenia is always present and, in severe cases, is less than 30 x 109/L. The bone marrow shows hypoplasia, with loss of hematopoietic tissue. with >70% yellow marrow  TREATMENT Medications as immunosuppressants Blood transfusions Bone marrow transplant.  Currently, the most effective therapy for AA is hematopoietic stem cell transplantation; however, 10 Female Male Response on steroid (+) Response on treatment (-) Pre-B-ALL Hypoploid, t(9;22)/t(9;11) Hyperploid FAB L2/L3 FAB L1 ↑↑LDH high ↑LDH moderate visceromegaly Prognostic factors are those measurements available at the time of diagnosis that are associated with disease-free or overall survival and can often be used to predict the natural history of the tumor. Optimizing treatment based on prognostic factors plays an important role in the management of cancer. Visceromegaly is enlargement of the organs inside the abdomen, such as the liver, spleen, stomach, kidneys, or pancreas Hyperploid A chromosomal abnormality in that is characterized by an addition of chromosomes that results in a chromosome number that is not an exact multiple of the haploid number.  Malignant Lymphoma Parameter Hodgkin Lymphoma Non-Hodgkin Lymphoma Distribution Usually central nodes Usually involves peripheral nodes Extranodal disease Uncommon Common Cell type Abnormal bizarre cells Resemble normal lymphoid cells Reed-Sternberg cell Present Absent Lymphomas are a group of neoplastic diseases caused by malignant lymphocytes that accumulate in lymph nodes and other lymphoid tissue and cause the characteristic clinical feature of lymphadenopathy. Occasionally, clonal lymphocytes may spill over into blood (‘leukaemic phase’) or infiltrate organs outside the lymphoid tissue. The major subdivision of lymphomas is into Hodgkin lymphoma (HL) and non-Hodgkin lymphoma (NHL), and this is based on the histological presence of Reed–Sternberg (RS) cells in Hodgkin lymphoma. Secondary or peripheral lymphoid organs, which include lymph nodes and the spleen  Reed-Sternberg cell  Tumor cell found in Hodgkin lymphoma.  Has two or more nuclear lobes containing inclusion-like nucleoli giving the appearance of owl's eye. Reed–Sternberg cells (also known as lacunar histiocytes for certain types) are different giant cells found with light microscopy in biopsies from individuals with Hodgkin's lymphoma (a.k.a. Hodgkin's disease, a type of lymphoma). RS cell is of B-lymphoid lineage and that it is often derived from a B cell with a ‘crippled’ immunoglobulin gene caused by the acquisition of mutations that prevent synthesis of full- length immunoglobulin. Human leukocyte antigen (HLA) class I expression is usually lost on the neoplastic cells and mutation of the β2-microglobulin gene is frequent. The Epstein–Barr virus (EBV) genome has been detected in over 50% of cases in Hodgkin tissue, but its exact role in the pathogenesis is unclear Staging of Hodgkin lymphoma Stage I indicates node involvement in one lymph node area. Stage II indicates disease involving two or more lymph nodal areas confined to one side of the diaphragm. Stage III indicates disease involving lymph nodes above and below the diaphragm. Splenic disease is included in stage III, but this has special significance. Stage IV indicates involvement outside the lymph node areas and refers to diffuse or disseminated disease (refers to a diffuse disease-process, generally either infectious or neoplastic.) in the bone marrow, liver and other extranodal sites. Non-Hodgkin B-cell Lymphoma The neoplastic B- More common than cell may express Non-Hodgkin T-cell CD20 on their lymphoma. surface. May be: Indolent ( slow to grow) Aggressive Highly aggressive  The non-Hodgkin lymphomas (NHL) are a large group of clonal lymphoid tumours, about 85% of B cell and 15% of T or NK (natural killer) cell origin (Table 20.1). Their clinical presentation and natural history are much more variable than those of Hodgkin lymphoma. NHL are characterized by an irregular pattern of spread and a significant proportion of patients develop disease outside the lymph nodes. Their frequency has increased markedly over the last 50 years and, with an incidence of approximately 17 in 100 000 in the UK, they now represent the fifth most common malignancy in some developed countries Indolent lymphomas advance more slowly than aggressive lymphomas, and they often don’t cause apparent symptoms early on. Many indolent lymphomas respond well to treatment, but they are usually challenging remove completely. Aggressive lymphomas advance more quickly than indolent lymphomas. Patients with aggressive lymphomas often develop symptoms sooner and require treatment immediately after their diagnosis. Based on the subtype of lymphoma, symptoms may vary from enlarged lymph nodes and weight loss to broader bone and skin issues. That said, aggressive lymphomas tend to respond well to cancer treatments. Proposed cellular origin of B-lymphoid malignancies  Normal B cells migrate from the bone marrow and enter secondary lymphoid tissue. When they encounter antigen, a germinal centre is formed and B cells undergo somatic hypermutation of the immunoglobulin (Ig) genes (is a process in which point mutations accumulate in the antibody V-regions of both the heavy and light chains, at rates that are about 106-fold higher than the background mutation rates observed in other genes). Finally, B cells exit the lymph node as memory B cells or plasma cells. The cellular origin of the different lymphoid malignancies can be inferred from immunoglobulin gene rearrangement status and membrane phenotype. Mantle cell lymphoma and a proportion of B-cell chronic lymphocytic leukaemia (B-CLL) cases have unmutated immunoglobulin genes whereas marginal zone lymphoma, diffuse large cell lymphoma, follicle cell lymphoma, lymphoplasmacytoid lymphoma and some B-CLL cases have mutated immunoglobulin genes  Types Diffuse large B-cell lymphoma Follicular lymphoma Burkit lymphoma Mantle lymphoma Marginal zone lymphoma Lymphoplasmacytic lymphoma  Diffuse large B-cell lymphoma (DLBCL) The most common type of Non-Hodgkin B-cell lymphoma. Aggressive. can develop in the lymph nodes or in “extranodal sites” (areas outside the lymph nodes) such as the gastrointestinal tract, testes, thyroid, skin, breast, bone, brain, or essentially any organ of the body. It may be localized (in one spot) or generalized (spread throughout the body). Despite being an aggressive lymphoma, DLBCL is considered potentially curable. Diffuse large B cell lymphoma (DLBCL) is a heterogeneous group of tumors composed of large B lymphoid cells diffuse large B cell lymphoma.  Diffuse large B cell lymphoma. Abnormal infiltrate composed of large lymphoid cells with pale-staining, vesicular chromatin (numerous clear areas resulting from the irregular distribution of chromatin, creating the impression of many small sacs or vesicles), irregular nuclear outlines, and basophilic nucleoli. Several mitotic figures are present  Follicular lymphoma Indolent lymphoma. Results from chromosomal translocation (14,18). BCL-2 gene is placed after immunoglobulin heavy chain promoter (IgH) on chromosome 14. BCL-2 inhibits cell death.  causing little or no pain. Follicular lymphoma (FL) is the second most common type of non-Hodgkin lymphoma (NHL) and accounts for almost 30% of all lymphomas. Follicular lymphoma (FL), an indolent neoplasm caused by a t(14;18) chromosomal translocation that juxtaposes the BCL2 gene and immunoglobulin locus, has a variable clinical course and frequently undergoes transformation to an aggressive lymphoma. BCL2 encodes a protein that inhibits apoptosis. In general, follicular (nodular) NHL has worse prognostic compared to diffuse NHL  Burkitt lymphoma  Highly aggressive.  Chromosomal translocation (8,14).  MYC gene is placed after IgH promoter.  MYC gene activates cell metabolism and growth leading to cell division. Burkitt lymphoma often presents with disease involving the intestine, ovaries, or kidney.  Burkitt lymphoma is considered the most aggressive form of lymphoma and is one of the fastest growing of all cancers. But it is very rare, accounting for about 2 percent of all lymphoma diagnoses. The disease originates in mature B-lymphocytes, is most often diagnosed in young adults and children, especially male. Burkitt lymphoma is a high-grade NHL with a high incidence in Africa (endemic subtype). It represents approximately one-third of all pediatric lymphomas occurring outside Africa (sporadic Burkitt lymphoma). Many adult cases occur in immunocompromised individuals such as those infected with the HIV virus. Burkitt lymphoma often involves extranodal sites The presence of the MYC-associated translocation [t(8;14) MYC/Immunoglobulin heavy chain gene (IGH)] or variants is necessary to confirm all but the most classic cases.  Burkitt lymphoma In Africa:  Extranodal involvement of the Neoplastic B-cell Jaw.  Associated with Epstein-Barr virus (EBV). Tingle body Outside Africa:  Extranodal involvement of Abdomen (ileocecal junction). Starry sky appearance  Less associated with EBV.  A biopsy of Burkitt lymphoma usually reveals a diffuse infiltrate of neoplastic cells demonstrating a “starry sky” appearance (Figure 28-8). The “sky” represents the blue nuclei of the neoplastic lymphocytes; the “stars” are formed by scattered pale-staining tingible body macrophages. The infiltrating lymphoid cells are intermediate in size with nuclei approximately the same size as those of the tingible body macrophages. Multiple small nucleoli are usually present, and mitotic figures and apoptotic bodies are frequent. The latter two features are characteristic of high-grade lymphoma A tingible body macrophage. A tingible body macrophage (TBMs) is a type of macrophage predominantly found in germinal centers, containing many phagocytized, apoptotic cells in various states of degradation, referred to as tingible bodies (tingible meaning stainable). Tingible body macrophages contain condensed chromatin fragments. The ileocecal junction marks the transition from the small bowel to the large bowel.  Mantle lymphoma Aggressive. Results from chromosomal translocation(14,11). BCL-1 gene is placed next to Ig promoter. BCL-1 encodes the protein Cyclin D1 which stimulates cell growth and division. outer edge of a lymph node follicle (the mantle zone)  Mantle-cell lymphoma comprises 2%–10% of all non-Hodgkin's lymphomas (NHLs). Patients present with generalized disease, and have a poor prognosis. Three different histologic patterns (mantle zone, nodular, and diffuse) Mantle cell lymphoma usually presents with disseminated disease involving multiple lymph node groups, bone marrow, peripheral blood, spleen, liver, and gastrointestinal tract. Gastrointestinal tract involvement can present as multiple polyps involving the small bowel The mantle zone (or just mantle) of a lymphatic nodule (or lymphatic follicle) is an outer ring of small lymphocytes surrounding a germinal center. MCL cells generally over-express cyclin D1 due to a t(11:14) chromosomal translocation in the DNA. Mantle cell lymphoma usually presents with disseminated disease involving multiple lymph node groups, bone marrow, peripheral blood, spleen, liver, and gastrointestinal tract. Gastrointestinal tract involvement can present as multiple polyps involving the small bowel   Indolent lymphoma.  The most common type of Marginal Zone lymphoma is Mucosa-Associate-lymphoid-tissue (MALT). Marginal zone  Also, there is Nodal marginal zone lymphoma (lymph nodes, spleen). lymphoma  t(11;18)(q21;q21) translocation-an API2-MALT1 fusion proteinprotein-promotes the continuous activation of a transcription factor, NF-κB  Marginal zone B cells (8% of cases ) are innate lymphoid cells that normally function by rapidly mounting IgM antibody immune responses to antigens such as those presented by infectious agents and damaged tissues. They are lymphocytes of the B-cell line that originate and mature in secondary lymphoid follicles and then move to the marginal zones of mucosa-associated lymphoid tissue (i.e. MALT), This type occurs outside the lymph nodes in places such as the stomach, small intestine, salivary gland, thyroid, eyes, and lungs. MALT lymphoma is divided into two categories: gastric, which develops in the stomach and is the most common site, and nongastric, which develops outside of the stomach. In many cases of MALT lymphoma, the patient has a previous medical history of chronic infection, inflammation, or autoimmune disorders at the affected organ. API2 gene with a part of the MALT1 gene to create a fusion gene that encodes an Api2-Malt1 fusion protein-promotes the continuous activation of a transcription factor, NF-κB. NF-κB controls the expression of various genes which increase the survival, cytokine production, and other potentially malignant behaviors of cells.  Lymphoplasmacytic lymphoma (LPL) Indolent lymphoma. Involves BM, lymph nodes, and spleen. Neoplastic B-cell produces immunoglobulin (M-protein). M- protein is found at a high level in the blood. A high concentration of M- protein increases the viscosity of the blood and causes Waldenstrom Macroglobulinemia.  Lymphoplasmacytic lymphoma (LPL) is a neoplasm that primarily involves the bone marrow and sometimes the spleen and lymph nodes and is composed of a mixture of neoplastic small lymphocytes and plasma cells. Waldenström macroglobulinemia is the combination of LPL with bone marrow involvement and an IgM monoclonal paraprotein. is a type of cancer affecting two types of B cells: lymphoplasmacytoid cells and plasma cells. Both cell types are white blood cells. It is characterized by having high levels of a circulating antibody, immunoglobulin M (IgM), The most commonly associated mutations, based on whole-genome sequencing of 30 patients, are a somatic mutation in MYD88 (90% of patients) and a somatic mutation in CXCR4 (27% of patients) TREATMENT Treatment of symptomatic patients includes rituximab, which can be used in combination with chemotherapy. Autologous bone marrow transplant is a recent treatment option. Plasmapheresis to treat hyperviscosity is used to reduce paraprotein levels. The median survival for patients with LPL is typically 5–10 years  Non-Hodgkin T- cell Lymphoma Adult T-cell lymphoma (leukemia). Mycosis Fungoides.  Adult T-cell lymphoma (leukemia)  Abnormal leukocytes in the bloodstream.  Caused by Human T- lymphotropic Virus (HTLV).  HTLV spreads through the body fluids and infects T-cell leading to incorporate DNA and mutation.  Mycosis Fungoides T-cell lymphoma of the skin. Causes patches on the skin that looks a bit like a fungal infection. Neoplastic CD4+ helper cell looks like (cerebriform) nucleus like a brain. If these cells circulate in the blood, they will cause Sezary syndrome It generally affects the skin, but may progress internally over time. Symptoms include rash, tumors, skin lesions, and itchy skin. Sézary syndrome is a rare cancer that affects the skin and the blood. It can lead to: Cancerous cells in the blood. Enlarged lymph nodes. Extensive skin rashes or lesions (tumor or other skin abnormalities). Sézary syndrome is s an aggressive form of cutaneous T-cell lymphoma  Symptoms Cerebriform cell  Diagnosis Imaging studies like CT-Scan. Biopsy from lymph nodes.  Treatment Chemotherapy and Radiation. If CD20+ expression —> Rituximab. Stem cell transplant.  Transformed mycosis fungoides (T-MF) is often associated with the appearance of a CD20 component Cluster of differentiation 20 (CD20) is expressed in Reed Sternberg (RS) cells of 11-35% of classical Hodgkin lymphoma (cHL) cases with very controversial prognostic significance. Rituximab is not considered part of standard therapy in cHL but has shown promising responses in the relapsed setting. CD20 is a transmembrane protein, originally identified as a B- cell surface marker involved in Ca++ channeling, B-cell activation, and proliferation (Somasundaram et al., 2011; Tedder & Engel, 1994). Using gene expression profiling, CD20 has been identified as one of the top 22 genes in melanoma that defines the aggressive nature of the disease  MPO is the most sensitive and specific stain for granulocytes, which stain intensely. Monocytes stain less intensely than neutrophils. Lymphocytes do not exhibit MPO activity. Therefore, MPO is useful in differentiating acute myeloid leukemia (AML) from acute lymphoblastic leukemia (ALL) and subgrouping of the AMLs (Table 37-12, Table 37-13). A positive reaction is seen in myeloblasts, and weak staining is rarely seen in monoblasts. Leukemic blasts that are negative for MPO can represent lymphoblasts, AML (minimal differentiation), monoblasts, erythroblasts, and megakaryoblasts, or undifferentiated leukemia. The presence of many mature neutrophils with negative MPO staining can indicate an MPO deficiency Blasts of AML showing a positive myeloperoxidase stain (brown- black color in cytoplasm) (bone marrow; MPO stain; 1000* magnification). Because of its fat solubility, SBB also can stain marrow fat and some cytoplasmic vacuoles of Burkitt lymphoma cells.  Sudan black B (SBB) is a diazo dye that stains phospholipids, neutral fats, and sterols.70 Cellular components containing lipids stain brown-black (Figure 37-30). SBB stains phospholipids in membranes of primary and secondary granules of the granulocytic series. Auer rods have a rich phospholipid membrane that the SBB stain identifies; its results parallel those seen with the MPO stain in myeloblasts and monoblasts. Lymphoblasts do not stain with SBB. This stain is useful when fresh specimens are not available for the MPO stain and in unusual cases when myeloblasts have an acquired deficiency of MPO. Therefore, SBB is useful in differentiating AML from ALL. Because of its fat solubility, SBB also can stain marrow fat and some cytoplasmic vacuoles of Burkitt lymphoma cells. Blasts of AML showing a positive Sudan black B stain (brown-black color in cytoplasm) (bone marrow; SBB stain; 1000* magnification). LAP scores can be decreased in chronic myelocytic leukemia (CML), paroxysmal nocturnal hemoglobinuria, immune thrombocytopenia and sometimes myelodysplasia. The LAP score for CML patients in blast crisis or with concurrent infections can be increased.  Because this enzyme is found within the secondary granules of maturing granulocytes, 100 segmented neutrophils/bands are counted, and each cell is graded using a scale of 0-4+ according to the appearance and intensity of the precipitated dye. The number of cells counted in each grade is multiplied by that grade, and the products are summed to obtain a total LAP score. The range of normal scores is 13–130, although this could vary slightly in each laboratory. The range of possible values is 0–400. A score higher than 160 is generally considered increased and lower than 13 is considered decreased. The LAP scores can be increased in leukemoid reaction (infection, inflammation), polycythemia vera, pregnancy, newborns, stress, oral contraceptives, and medications (steroids, estrogen, lithium, growth factors).  Mature granulocytes (the intensity of staining increases with maturity), platelets, megakaryocytes, and monocytes are stained with periodic acid-Schiff. However, the early myeloid cells, the erythroid cells, and many of the lymphoid cells are negative with PAS. Therefore, PAS positivity in these cells can indicate abnormal glycogen metabolism and can be diagnostically important AML-diffuse  Leukocyte esterases Leukocyte esterase (LE) is an esterase (a type of enzyme) produced by leukocytes. There are 2 types :  Specific esterase naphthol AS-D chloroacetate esterase (CAE)  It is positive in the myeloid series. Positivity is in myeloblast, mature stages stain strongly. Positivity is confined to neutrophils series and mast cells.  Auer rods are positive  These identify the cells of the granulocytic series.  It does not stain lymphocytes and monocytes  negative  The reaction product is bright red.   Nonspecific esterase (NSE) α-naphthyl butyrate esterase (ANBE) or α-naphthyl acetate esterase (ANAE)  Activity is seen in monocytes  stain strongly.  It is positive in monocyte and monocyte precursors \ monoblast cell line  Megakaryocytes and platelets, histocyte, macrophages, lymphoblasts of ALL are positive  The cells of granulocytic series (granulocytes) are negative  The α-naphthyl butyrate stain more specific, while the α-naphthyl acetate stain is more sensitive.  The reaction product is red \ brown granules.. brick-red staining  Inhibition of nonspecific esterase by sodium fluoride NaF (Monocytic nonspecific esterase is Fluoride sensitive) is used to identify normal and leukemic mononuclear phagocytes cytochemically.  In a suspected myeloid leukemia: Acute myelomonocytic leukemia (AMML): Dual esterase stains (Dual esterase test)