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Hegbom and Edenberg Internal Medicine VI Hematology FE -Erik Hegbom"Of all the things I have lost, I miss my mind the most" -Andreas Edenberg"By the people who took a glance at the book, for the people who never will know what it looked like" 1 of 46 Hegbom and Edenberg Hematology 1. Haemopoiesis Ontogenesis Reticulocyte counting Hemopoiesis: Hemopoiesis includes both erythropoiesis and leukopoiesis, as well as thrombopoiesis. In the adult, erythrocytes, granulocytes, monocytes, and platelets are formed in the red bone marrow; lymphocytes are also formed in the red bone marrow and in the lymphatic tissues. Blood cells are derived from a common stem cell: The pluripotential stem cell, the cell has the appearance of a small to medium-sized lymphocyte, and is CD 34 +. Cytokines and growth factors influence and direct proliferation and maturation of stem cells into specific lineages. Erythrocytes develop form the multipotential myeloid stem cell (CFU-GEMM)

under the influence of erythropoietin, GM-GSF, and specific interleukins. The erythropoietin-sensitive erythrocyte progenitor cell CFU-E gives rise to the first recognizable erythrocyte precursor, the proerythroblast. The reticulocyte is the first cell in the erythroid lineage which has extruded its nucleus Kinetics: Nearly all erythrocytes are released into the circulation as soon as they are formed. Erythrocyte formation and release are regulated by erythropoietin, synthesized and secreted by the kidney in response to decreased blood concentration. Lymphopoiesis: Progeny of the multipotential lymphoid stem cells that are destined to become T cells leave the bone marrow and travel to the thymus, where they complete their differentiation. Cells destined to become B cells originate in bursa-equivalent organs such as the bone marrow, GALT, and spleen. Bone marrow is the primary site of lymphopoiesis Granulopoiesis, monocyte development, and megakaryocyte development. Ontogenesis:

Hemopoiesis in embryonic and fetal life(?, I guess so.) • • • Early developmental stages: Yolk sac 2nd trimester: liver, spleen to a very limited degree. 3rd trimester: bone marrow Hemopoiesis occurring in the yolk sac, spleen and liver is largely limited to erythroid cells, although some leukopoiesis occur in the liver. After birth, hemopoiesis takes place only in the red bone marrow and lymphatic tissues In infants, the hematopoietic bone marrow is found in practically all bones. In adults it is found in vertebrae, ribs, sternum, skull, sacrum and pelvis, and in the proximal parts of long bones. Reticulocyte counting: The reticulocyte count is ordered to determine if the bone marrow is responding adequately to the bodys need for RBCs and to help determine the cause of and classify different types of anemia. The number of reticulocytes must be compared to the number of erythrocytes. Reticulocytes can be distinguished from mature RBCs because they still contain remnants of RNA

Circulating reticulocytes generally lose their RNA in 1-2 days, thus becoming mature RBCs. In order to get a more accurate assessment the reticulocyte index can be used (corrects for anemia) Reticulocyte (%) = [number of reticulocyte / number of RBCs]*100 Reticulocyte index = reticulocyte count * [measured hct / normal hct] Manual method; stain with methylene blue or brilliant cresyl blue. Calculate reticulocytes per 1000 red cells Flow cytometer: Mature red blood cells are characterized by low intensity fluorescence signal, while reticulocytes because of their residual RNA display a higher value. Thiazol-orange is used as a dye 2 of 46 Hegbom and Edenberg 2. Essential normal values in haematology Peripheral blood smear, bone marrow specimen (laboratory method) Essential normal values in haematology: Check your book. Peripheral blood smear: The ideal PBS requires a fresh drop of capillary blood without anticoagulation. However, a fresh venous sample collected in EDTA is

satisfactory and as it is convenient is widely used. A drop of blood is placed on one end of a clean glass slide and is spread either mechanically or manually resulting in a tongue or bullet shaped smear covering 1/2-2/3 of the slide. The slide is rapidly air dried, fixed in methanol and stained typically with Wright-Giemsa. The examination starts with a macroscopic view to evaluate the quality of the smear based on overall appearance. The microscopic analysis begins on lower power (10x), primarily to assess cellular distribution, staining quality, and to select an area where the RBCs are barely touching each other. This area is used to conduct a complete assessment of the cellular elements on higher magnification On hi-dry (40x), the slide is principally scanned to obtain a WBC estimate. All of the detailed analysis of the cellular elements is performed using oil immersion. This final microscopic examination is performed at 50x or 100x oil immersion and includes: • • • • •

• A WBC differential The identification of abnormal or peculiar leukocytes Assessment of RBC morphology The number and morphology of the platelets The identification of intra- and extra-cellular elements Assessment of any organisms present Laboratories are now using automated hematology instruments to produce complete blood counts (CBC) that are accompanied by a multipart report detailing the following: • • • Hemogram: measured and calculated parameters Histograms: size distribution of WBC, RBC and PLT Cytogram: WBC differential To date, however, no instrument can completely replace the visual examination of blood cells. Bone marrow specimen: Special needles are used for both biopsies and aspirations. Complications from a bone marrow aspiration and biopsy include infection and bleeding. With proper sterile atraumatic technique these complications are rare. Absolute contraindications to performing the procedure include hemophilia or related coagulation disorders and

infection involving the biopsy area. Thrombocytopenia is not a contraindication, but take care to avoid trauma that is more significant than usual. Biopsies never should be obtained from the sternum; only aspiration is performed in that area The following are the safe and preferred sites for bone marrow aspiration and bone marrow biopsy: 1. Posterior iliac crest (both aspiration and biopsy) 2. Sternum (aspiration only in adults) 3. Anterior iliac crest (both aspiration and biopsy) Depending on the indication for the bone marrow aspiration and biopsy, the samples are sent for the appropriate tests, in addition to the routine Wright or Giemsa staining and histopathological processing of the biopsy samples. Examples of tests performed on biopsy and aspiration samples include flow cytometry, fluorescence-in situ-hybridization (FISH) studies, and cytogenetics when hematological malignancies and lymphoproliferative disorders are suspected; specialized cytochemistry when the above are

suspected; Prussian blue staining for iron when disorders of iron metabolism (sideroblastic anemias) are suspected; and fungal, acid-fast bacilli, and bacterial cultures when these conditions are suspected or a FUO is being investigated. 3 of 46 Hegbom and Edenberg 3. B12 and folic acid deficiency Characteristics of the megaloblastic bone marrow B12 deficiency: The vitamin is a cofactor for 2 reactions; for mehtionine synthetase in the conversion of homoCys to methionine, and for the conversion of methylmalonyl-CoA to succinyl-CoA. B12 is present in all foods of animal origin After being ingested, B12 is bound to IF, secreted by gastric parietal cells. This complex is absorbed in the terminal ileum The vitamin is bound to transcobalmin II in plasma. B12 is stored in the liver which contains 2000-5000µg The daily use and absorption is around 5µg. Causes of deficiency: Strict vegetarians, gastrectomy, resection of ileum, severe Crohns disease, parasites, pancreatic insufficiency.

The most common cause is associated with pernicious anemia – rare clinically before age 35 Symptoms and signs: • • • • Megaloblastic anemia. In advanced cases; Hct is 10-15%, leukopenia and thrombocytopenia Changes in mucosal cells glossitis, diarrhea. Neurological syndrome. First peripheral nerves are affected, causing parasthesis Next the posterior columns are affected, causing difficulty with balance. In more advanced cases, cerebral functions may be affected as well Patients are usually pale and may be mildly icteric. Lab.: • • • • • • Anemia. MCV↑, between 110 and 140 fL However MCV may be normal Peripheral smear, often abnormal; anisocytosis and poikilocytosis. A characteristic finding is the macro-ovalocyte Hypersegmented neutrophils (mean lobe count > 4). Reticulocyte count is reduced Bone marrow morphology: Erythroid hyperplasia. Megaloblastic changes in erythroid cells In the myeloid series, giant metamyelocytes are seen. Elevated serum LDH and

a modest increase in indirect bilirubin (intra medullary destruction of developing erythroid cells). Diagnosis is made by low vit. B12 serum level (< 170 pg/ml) Schilling test is rarely used. Treatment: vit. B12 orally or im Hypokalemia may complicate first several days of treatment Hematological picture normalizes in 2 months. CNS symptoms and signs are reversible if they are of relatively short duration (<6 months) Folic acid deficiency: Tetrahyrofolate is a mediator in one-carbon transfers. Folic acid is present in most fruits and vegetables Its absorbed along the entire GI tract. Causes of deficiency: Inadequate intake – alcoholics, anorectic patients, persons who do not eat fresh fruits and vegetables. Drugs – phenytoin, TMP-SMX, sulfazalazine. Increased requirement – chronic hemolytic anemia, pregnancy, exfoliative skin disease Symptoms and signs: • Megaloblastic anemia and megaloblastic changes in mucosa (see above). Lab.: • • As for B12 deficiency. Low

red blood cell folate level. Treatment: Folic acid, 1mg/d orally. 4 of 46 Hegbom and Edenberg 4. Iron deficiency anemia Prussian blue staining Iron deficiency anemia: Most common cause of anemia worldwide. Total body iron ranges between 2-4 gram, most of which is present in hemoglobin. Absorption of iron occurs in the stomach, duodenum, and upper jejunum In general, iron metabolism is balanced between absorption of 1mg/d and loss of 1mg/d. Causes: Diet, decreased absorption, pregnancy, menstruation, lactation, hemoglobinuria, GI bleeding, etc. Symptoms and signs: • • • Anemia (fatigue, tachycardia, palpitations, tachypnea on exertion). Severe deficiency; smooth tongue, brittle nails, cheilosis, dysphagia (esophageal webs). Many develop craving for specific food. Lab.: • • • First – anemia, low serum ferritin, TIBC rises. Later – After depletion of stores, RBC production continue with deficient supply of iron – MCV↓, serum iron↓, hypochromic, microcytic

cells seen on peripheral smear. With further progression anisocytosis and poikilocytosis develop. Treatment: Identification of cause. Oral iron; ferrous sulfate, 325 mg three times daily An appropriate response is full return of Hct after 2 months. Iron therapy should continue for 3-6, months after restoration of normal hematologic values Parenteral iron; sodium ferric gluconate. Indications are intolerance to oral iron, refractoriness to oral iron Because of the possibility of anaphylactic reactions, parenteral iron should only be used in cases of persistent anemia after a reasonable course of oral therapy. Prussian blue staining: Prussian blue reaction - PRINCIPLE: The reaction occurs with the treatment of sections in acid solutions of ferrocyanides. Any ferric ion (+3) in the tissue combines with the ferrocyanide and results in the formation of a bright blue pigment called Prussian blue" or ferric ferrocyanide. Fast red is used as a nuclear stain RESULTS: Iron (hemosiderin)

– blue Nucleus – red Background – pink. 5 of 46 Hegbom and Edenberg 5. Vitamin B6 deficiency, refractory anemia with ringed sidereoblasts Vitamin B6, principally in the form of the coenzyme pyridoxal 5-phosphate, is involved in a wide range of biochemical reactions, including the metabolism of amino acids and glycogen, the synthesis of nucleic acids, hemoglobin, sphingomyelin and other sphingolipids, and the synthesis of the neurotransmitters serotonin, dopamine, norepinephrine and gamma-aminobutyric acid (GABA). Pyridoxine is needed for niacin production. Homocystine metabolism is dependent on pyridoxine, and high homocystine levels can result from pyridoxine deficiency. Pyridoxine is widespread in foods Pyridoxine deficiency causes blood, skin, and nerve changes. This vitamin is unique in that both deficiency and excess can cause peripheral neuropathy. The classical symptoms and signs of vitamin B6 deficiency are a microcytic, hypochromic anemia, seizure activity,

seborrheic dermatitis, confusion and depression. Vitamin B6-deficiency states in infants and children primarily result in electroencephalogram abnormalities and seizure activity, while in adults; vitamin B6 deficiency primarily results in cheilosis, glossitis, stomatitis, anemia, irritability, confusion and depression. Vitamin B6 deficiency may result from the use of certain drugs, including isoniazid (isonicotinic acid hydrazide or INH), penicillamine, cycloserine, ethionamide, hydralazine and theophylline. Subclinical vitamin B6 deficiency frequently occurs in those with malabsorption syndromes, uremia, cancer, heart failure and cirrhosis, and in alcoholics, the elderly and adolescent females and during pregnancy. In the elderly and in those with malabsorption syndromes, clinical deficiency of the vitamin may occur Lab.: Serum pyridoxal 5-phosphate (PLP) Treatment: Supplementation of pyridoxine hydrochloride. Refractory anemia with ringed sideroblasts: Sideroblastic anemias are a

heterogeneous group of disorders in which Hb synthesis is reduced because of failure to incorporate heme in protoporphyrin. Instead iron accumulates in mitochondria ringing the red cell nucleus forming "ringed sideroblasts". Most sideroblastic anemias are acquired and are associated with ethanol abuse, VitB6 antagonists, lead poisoning, and myelodysplasia. The hereditary form (rare) is an X-linked recessive trait with variable expression Hereditary sideroblastic anemias are usually microcytic/hypochromic and thus must be distinguished from iron deficiency and thalassemia. Symptoms: Those of anemia. Lab.: • • • • Hct↓, MCV may be normal, slightly increased, or low. Peripheral blood smear shows a dimorphic population of RBCs, one normal and one hypochromic. High serum iron and high transferrin saturation. Diagnosis is made by examination of bone marrow: Marked erythroid hyperplasia. Iron stain shows presence of ringed sideroblasts. Treatment: Underlying cause.

Hereditary sideroblastic anemia responds somewhat to vitB6 6 of 46 Hegbom and Edenberg 6. Sphaerocytosis G-6-PHD deficiency Reticulocyte counting (theoretical, practical) Hereditary sphaerocytosis: AD disease of variable severity. The membrane defect in hereditary spherocytosis is an abnormality in spectrin (membrane protein). The result is a decrease in the surface-to-volume ratio that results in a spherical shape of the cell These RBCs are unable to pass through the fenestrations in the splenic red pulp. Hemolysis takes place in the spleen Symptoms and signs: Anemia may or may not be present (bone marrow compensation). Severe anemia may occur in infections, and folic acid deficiency. Symptoms of chronic hemolysis Patient may be icteric and have a palpable spleen Lab.: • • • • • • Anemia of variable severity. Reticulocytosis is always present. Sphaerocytes on peripheral smear (usually only a small % of RBCs). Microcytosis and increased MCHC. Positive osmotic

fragility test. LDH and indirect bilirubin↑, low haptoglobin. Treatment: Uninterrupted supplementation with folic acid, 1mg/d. The treatment of choice is splenectomy Glucose-6-phosphate dehydrogenase deficiency: The hexose monophosphate shunt is needed for generating reduced glutathione, which protects hemoglobin from oxidative denaturation. In the absence of reduced glutathione, hemoglobin may become oxidized The ox Hb. then denatures and form precipitants called Heinz bodies Heinz bodies cause membrane damage, which leads to removal of these cells by the spleen. G6PD def is an X-linked disorder commonly affecting black men Symptoms and signs: Hemolysis occurs as a result of oxidative stress – infection or drugs. Common drugs initiating hemolysis are dapsone, primaquine, quinidine, quinine, sulfonamides, and nitrafurantoin. The hemolytic episode is self-limited Lab.: Normal blood between episodes During episodes of hemolysis, there is reticulocytosis and increased serum indirect

bilirubin Heinz bodies may be demonstrated by staining peripheral blood with cresyl violet. Enzyme assays Treatment: Avoid precipitants. Reticulocyte counting: Topic 1 7 of 46 Hegbom and Edenberg 7. Haemoglobinopathies The thalassemias are due to reduced globin chain synthesis causing decreased Hb synthesis hypochromic microcytic anemia. The normal adult hemoglobin is HbA (α2β2). The α gene is found in 2 copies on chr 16 The β gene is found on chr 11 A.) α-thalassemias: Inherited disease seen mostly in persons from southeast Asia Primarily due to gene deletions causing reduced αglobin synthesis There is no change in the % distribution of HbA, A2, and F In severe forms β chains may form a β4 tetramer called HbH, or, in infancy, γ4 tetramer (Bart’s Hb) is formed. Symptoms and signs: • • • • Silent carrier; when 3 α-globin chains are present the patient is hematological normal. Thalassemia minor/α-thalassemia trait; 2 α-globin genes are present, mild

microcytic anemia. Hemoglobin H disease; 1 α-globin chain present, chronic hemolytic anemia of variable severity. Hydrops fetalis; all 4 α-globin chains are deleted, fetus is stillborn. Lab.: • • α-Thalassemia trait; mild anemia, with low MCV. Peripheral blood shows microcytes, hypochromia, target cell, acanthocytes Reticulocyte count and iron levels are normal. Hemoglobin H disease; more marked anemia (hct. 22-32%), MCV is low Peripheral smear is markedly abnormal Reticulocyte count is elevated. Electrophoresis will show presence of HbH Treatment: Patients with HbH disease should take folate supplementation and avoid medicinal iron and oxidative drugs. Transfusions may be necessary. B.) β-thalassemias: Autosomal inheritance Seen mostly in persons of Mediterranean origin Caused by point mutations causing decreased ( β+) or absent (β0) β-chain synthesis. There is a relative increase in Hb A2 and Hb F compared to HBA In the presence of reduced β chains, the excess α

chains precipitate. This leads to intramedullary and peripheral hemolysis The bone marrow becomes hyperplastic, erythropoiesis is ineffective. Bone deformities and pathologic fractures may be seen Symptoms and signs: • • • β-Thalassemia major; persons homozygous for β-thalassemia. Normal at birth but after 6 months severe anemia requiring transfusions develop. Growth failure, bone abnormalities, jaundice, and hepatosplenomegaly Thalassemia intermedia; homozygous for a milder form. Chronic hemolytic anemia, require transfusions under stress Hepatosplenomegaly and bone deformities. Thalassemia minor; heterozygous for β-thalassemia. Clinically insignificant hemolytic anemia Lab.: • • β-thalassemia minor; modest anemia, MCV decreased, microcytosis, hypochromia. Β-thalassemia major; severe anemia. Bizarre peripheral blood smear HbF is major Hb Treatment: Transfusions, folate, deferoxamine. Splenectomy may be performed Hemosiderosis is a serious problem Allogenic bone

marrow transplantations has become treatment of choice for β-thalassemia major. Sickle cell anemia: AR disorder leading to chronic hemolytic anemia. HbS gene is carried in 8% of American blacks In Hb S, valine is substituted for glutamic acid in the 6th amino acid of the β chain. When in the deoxy form, Hb S forms polymers that damage the RBC membrane. Distorted, inflexible RBCs adhere to vascular endothelium and plug small arterioles and capillaries, which leads to infarction. Venous plugging predisposes to thromboses Because sickled RBCs are fragile, the mechanical trauma of circulation produces hemolysis. Chronic compensatory marrow hyperactivity deforms the bones Hb F cannot participate in polymer formation and its presence retards sickling. Symptoms and signs: Most symptoms occur in homozygotes. Jaundice, anemia, pigment gallstones Hepatomegaly, but spleen not palpable in adults. Acute painful episodes due to vaso-occlusive crises may occur spontaneously or be provoked by

infection, dehydration, or hypoxia. Repeated episodes of vascular occlusion affect a large number of organs, especially the heart and liver, but also bones, spleen, lungs, brain. Patients have an increased risk of infections, especially pneumococcal and Salmonella infections Death from organ failure. Life expectancy 40-50 years of age Lab.: Chronic hemolytic anemia Sickled cells comprising 5-50% of red cells Reticulocytosis The WBC count is characteristically elevated. Elphoresis to detect HbS Treatment: Folic acid supplementation. Transfusions for aplastic or hemolytic crisis Precipitants of acute painful episodes should be avoided. Hydroxyurea, by increasing fetal Hb and thereby reducing sickling, decreases painful crises (by 50%) and transfusion requirements. Hb C disease: Homozygous HbC disease produce a mild hemolytic anemia with splenomegaly, mild jaundice, and pigment gallstones. The peripheral smear show generalized red cell targeting. 8 of 46 Hegbom and Edenberg 8.

Paroxysmal nocturnal hemoglobinuria PNH is now known to be a consequence of nonmalignant clonal expansion of one or several hematopoietic stem cells that are deficient in GPI-anchor protein (GPI-AP) acquired through a somatic mutation of PIG-A. • Recent information has led us to understand that PNH is not a monoclonal disease with a malignant phenotype. Rather, the clinical pathology may actually be an epiphenomenon resulting from an adaptive response to injury such as an immune attack on the stem cells of hematopoiesis. • In PNH, the peripheral blood and bone marrow is a mosaic composed of GPI-AP+ and GPI-AP- cells; with the GPI-AP-, cells can be derived from multiple mutant stem cells. The GPI-AP- mutant cells may appear to dominate hematopoiesis in PNH by providing a proliferative advantage under some pathological conditions. For example, if damage to stem cells causing bone marrow failure is mediated through a GPI-linked surface molecule, the PNH cells lacking these

molecules will survive. The close association with aplastic anemia and MDS suggests that the selection process arises as a consequence of this specific type of BM injury. Deficient hematopoiesis may occur due to diminished blood cell production with a hypoplastic bone marrow. Symptoms and signs: Episodic hemoglobinuria (most freq. episodes last 3-10 days) Dark urine in the morning Prone to thrombosis (This thrombosis is secondary to a lack of CD59 on platelet membranes that induces platelet aggregation and is highly thrombogenic, particularly in the venous system.), especially in hepatic and mesenteric veins May progress to either aplastic anemia, myelodysplasia, or to AML. Lab.: Anemia, reticulocytosis may be seen Pancytopenia can be seen Urine hemosiderin Serum LDH↑ Low serum haptoglobin Iron deficiency. Flow cytometry may confirm diagnosis by demonstrating absence of complement-regulating proteins Treatment: Iron and folic acid replacement. Prednisone is for unclear reasons

effective in decreasing hemolysis Allogenic bone marrow transplantation. Eculizumab is a potential future drug targeting terminal protein C5, 9 of 46 Hegbom and Edenberg 9.Autoimmune hemolytic anemia (primary, secondary) Warm antibody hemolytic disorder is an acquired disorder in which an IgG autoantibody is formed that binds to the RBC membrane. The autoantibodies are recognized by macrophages in the spleen and other portions of the RES. This interaction cause the formation of spherocytes. These spherocytes become trapped in the spleen The hemolysis is often severe and can be fatal Autoantibodies may arise spontaneously, or associated with some diseases as SLE, CLL, lymphomas. Some drugs also form autoantibodies against RBCs, either directly against membrane Ag, or against a hapten complex. Cold agglutinin disease (cold antibody disease) is caused by autoantibodies (IgM) that react at temperatures < 37° C and subsequently fixing complement. When the RBCs returns to warmer

temperature, the IgM dissociates, leaving complement on the cell The disease sometimes occurs with infections (especially mycoplasmal pneumonias or infectious mononucleosis) and lymphoproliferative states; about 50% of cases are idiopathic, which is the common form in older adults. Infections tend to cause acute disease, whereas idiopathic disease tends to be chronic. Hemolysis occur (rarely) mainly in the extravascular mononuclear phagocytic system of the liver The higher the temperature at which these autoantibodies react, the greater the hemolysis. Symptoms and signs: Warm antibody hemolytic anemia; anemia of rapid onset. Fever, angina, CHF may be seen Jaundice and splenomegaly are usually present. Cold agglutinin disease: Symptoms related to RBC agglutination on exposure to cold – numb fingers and toes. Lab.: • AIHA is suspected in patients with hemolytic anemia, particularly if symptoms are severe or other suggestive symptoms are present. Routine laboratory tests generally

suggest extravascular hemolysis (eg, hemosiderinuria is absent; haptoglobin levels are near normal) unless anemia is sudden and severe. Spherocytosis and a high MCHC are typical • AIHA is diagnosed by the direct Coombs test (direct antiglobulin test): Antiglobulin serum is added to washed RBCs from the patient; agglutination indicates the presence of immunoglobulin, generally IgG, or C3 bound to the RBCs. • Cold agglutinin disease will have a Coombs test positive for complement only. Treatment: Warm antibody disease: Prednisone is first choice. In drug induced disease the causative drug should be withdrawn, prednisone is not effective; rather infusion of Igs should be tried. If prednisone is ineffective splenectomy should be performed. Refractory disease may be treated with rituximab, danazol, immunosupressives, or intravenous immune globulin. Cold agglutination disease: Avoid exposure to cold. Patients with severe involvement may be treated with cyclophosphamide or cyclosporine.

10 of 46 Hegbom and Edenberg 10. Aplastic anemia Aplastic anemia is a condition of bone marrow failure that arises from injury to or abnormal expression of the stem cell. The bone marrow becomes hypoplastic, pacytopenia develops. Causes: The most common cause is autoimmune suppression of hemopoiesis by a T-cell mediated mechanism. Other causes are radio/chemotherapy, toxins (e.g benzene), drugs (eg chloramphenicol, anticonvulsants), SLE, PNH, pregnancy, congenital (Fanconi anemia). Symptoms and signs: • • Consequences of anemia, neutropenia, thrombocytopenia. Phys.ex may reveal pallor, purpura, and petechiae Presence of hepatosplenomegaly, lymphadenopathy, or bone tenderness should lead one to question the diagnosis. Lab.: • • Hallmark is pancytopenia. However early in the course of the disease, only one or two cell lines may be affected Hypocellular bone marrow biopsy with no abnormal cells. Treatment: In mild disease RBC and platelet transfusions are given as

necessary, and antibiotics are used to treat infections. Severe aplastic anemia – a.) for people less than 50 years who have HLA-matched siblings the treatment of choice is bone marrow transplantation. b) for people over 50 years or those without HLA-matched siblings, the treatment of choice is immunosuppression with antithymocyte globulin plus cyclosporine. Alternative drugs are cyclophosphamide and androgens. Course and prognosis: Allogenic bone marrow transplantation highly successful in children and adults with HLA-matched siblings. Durable complete response rate exceeds 80%. ATG treatment leads to partial response in around 60% of adults, and long-term prognosis of responders seems to be good. 11 of 46 Hegbom and Edenberg 11. Neutropenia (agranulocytosis) Neutropenia is present when the neutrophil count is below 1500/µL. The patient is increasingly vulnerable to infections by G + and G ÷ bacteria and by fungi. Severity of neutropenia relates to the relative risk of

infection When the neutrophil counts fall to <500/µL, endogenous microbial flora can cause infections. If counts fall to <200/µL, inflammatory response may be nonexistent The most common infections are septicemia, cellulitis, and pneumonia. The most common bacterial causes are coagulase-neg staphylococci and Staphylococcus aureus. Etiology: Acute neutropenia (occurring over hours to days) can develop from rapid neutrophil utilization or destruction, or from impaired production. Chronic neutropenia (lasting months to years) usually arises from reduced production or excessive splenic sequestration. Neutropenia may be classified as due to an (1) intrinsic defect in marrow myeloid cells or as (2) secondary due to factors extrinsic to marrow myeloid cells. Causes of (1)-This type of neutropenia is uncommon. Examples are Kostmann syndrome, aplastic anemia, cyclic neutropenia, myelodysplasia, chronic idiopathic neutropenia. Causes of (2)- Alcoholism, autoimmune, bone marrow

replacement by cancer, infections, cytotoxic chemotherapy or radiation, folate or B12 deficiency, hypersplenism., drugs (eg anticonvulsants, phenothiazines, penicillin) Symptoms and signs: Neutropenia is asymptomatic until infection develops. Fever is often the only indication of infection Focal symptoms may develop but are often subtle. Patients with drug-induced neutropenia due to hypersensitivity may have a fever, rash, and lymphadenopathy from the hypersensitivity. Diagnosis: Suspected in patients with frequent, severe, or unusual infections or in patients at risk. Confirmation is by CBC with differential. If acute neutropenia – At least 2 sets of bacterial and fungal blood cultures should be taken. Skin lesions are aspirated or biopsied for cytology and culture. Urinanalysis, urine cultures, and chest X-ray are obtained in all patients The most important test is bone marrow examination, which determines whether neutropenia is due to decreased marrow production or is secondary to

increased destruction or utilization of the cells (determined by normal or increased production of the cells). The bone marrow examination may also indicate the specific cause of neutropenia. Treatment: Acute neutropenia – Antibiotics if infection is suspected. Myeloid growth factors Potential offending drugs are stopped Chronic neutropenia – G-CSF, antibiotics if needed. For patients with autoimmune disorders or who have had an organ transplant, cyclosporine can also be beneficial. 12 of 46 Hegbom and Edenberg 12. Chronic neutropenia Differential diagnosis in eosinophilia Chronic neutropenia: See also previous topic. Causes: e.g • Kostmann syndrome and chronic benign neutropenia. • Chronic autoimmune neutropenia: This disorder is observed in adults and has no age predilection. As many as 36% of patients will exhibit serum antineutrophil antibodies, and the clinical course usually is less severe. Can be associated with other autoimmune diseases. Treatment with

corticosteroids • Nutritional deficiencies include vitamin B-12, folate, and copper deficiency • Splenic sequestration: The degree of neutropenia resulting from this process is proportional to the severity of splenomegaly and the bone marrows ability to compensate for the reduction in circulating bands and neutrophils Differential diagnosis of eosinophilia: In eosinophilia the peripheral eosinophil count is >450/µL. Primary eosinophilia: • • • Can be part of CML, AML, ALL, myelodysplastic syndromes. Chronic eosinophil leukemia (rare). Idiopathic hypereosinophilic syndrome. Only some patients with prolonged eosinophilia develop idiopathic hypereosinophilic syndrome. Although any organ may be involved, the heart, lungs, spleen, skin, and nervous system are typically affected The diagnosis is suspected in patients with eosinophilia without an obvious cause and with symptoms suggesting organ dysfunction. Differentiated by the above by being non clonal. Secondary

eosinophilia: • • • • • • In the Western world allergic and atopic diseases are the most common causes. Parasitic infestations, however protozoa and non-tissue invasive metazoa usually do not cause eosinophilia. Sometimes nonparasitic infections – e.g mycobacterial disease, infectious mononucleosis Tumors – Carcinomas, sarcomas, Hodgkins disease, non-Hodgkins lymphoma. Vasculitis, granulomatous, CT diseases. Adrenal hypoplasia, cirrhosis. 13 of 46 Hegbom and Edenberg 13. AML WHO classification Clinical signs Therapy Prognosis Bone marrow specimen In acute myelogenous leukemia the malignant cell loses ability to mature and differentiate. These cells proliferate and replace the normal bone marrow. Most of the clinical findings in acute leukemias are due to replacement of marrow elements. Less common manifestations result from organ infiltration. AML is chiefly an adult disease with a median age at presentation of 60 years. However this disease affects all age

groups Causes: • • • • • • Most patients who present with de novo AML have no identifiable risk factor. Antecedent hematologic disorders – The most common is MDS. Other include aplastic anemia, myelofibrosis, paroxysmal nocturnal hemoglobinuria, and polycythemia vera. Some congenital disorders have increased risk; e.g Downs syndrome, Fanconi anemia, congenital neutropenia Familial cases. Radiation and some toxins (benzene) are leukomogenic In addition, a number of chemotherapeutic agents may cause leukemia. The leukemias seen after toxins or chemotherapeutic exposure often develop from a myelodysplastic prodrome and are associated with abnormalities in chromosomes 5 and 7. Symptoms and signs: Most patients have been ill for only days or weeks. Bleeding (usually due to thrombocytopenia) occurs in the skin and mucosal surfaces. Less commonly, widespread bleeding in patients with DIC Infection is due to neutropenia Patients may also seek medical attention because of gum

hypertrophy and bone and joint pain. Patients with markedly elevated WBC counts (>100,000 cells/ L) can present with symptoms of leukostasis. Leukostasis is a medical emergency On examination, patients appear pale and have purpura and petechia. Stomatitis and gum hypertrophy may be seen in patients with monocytic subtypes of leukemia, as may rectal fissures. There is variable enlargement of liver, spleen, and lymph nodes Fever and other signs of infection can be present. Bone tenderness may be present Signs relating to leukostasis include respiratory distress and altered mental status. Lab.: • • • • • • • CBC with differential demonstrates anemia and thrombocytopenia to varying degrees. Patients with acute myelogenous leukemia (AML) can have high, normal, or low WBC counts. If DIC is present – elevated prothrombin time, decreased fibrinogen level, presence of fibrin split products. Peripheral blood smear: Blasts are usually seen. Schistocytes may be seen if DIC is

present Elevated serum LDH. Hyperuricemia may be seen Bone marrow aspiration: Usually hypercellular and dominated by blasts. More than 20 % blasts are required to make a diagnosis of acute leukemia. Blasts stain for myeloperoxidase Auer rods may be seen in myeloblastic and monoblastic cells. Flow cytometry, immunohistochemistry, cytology are used to diff. from ALL and to subtype AML Classification: Fab classification, traditional classification. (In your book) Acute myeloid leukemia, WHO classification: Acute myeloid leukemias with recurrent cytogenic translocations AML with t(8;21)(q22;q22), AML1 (CBFα)/ETO Acute promyelocytic leukemia (AML with t(15;17)(q22;q11-12) and variants, PML/RAXα) AML with abnormal bone marrow eosinophils (inv(16)(p13q22), CBFβ/MYH11X) AML with 11q23 (MLL) abnormalities Acute myeloid leukemia with multilineage dysplasia with prior myelodysplastic syndrome without prior myelodysplastic syndrome Acute myeloid leukemia and myelodysplastic syndrome, therapy

related Alkylating agent related Epipodophyllotoxin related (some may be lymphoid) Other types Acute myeloid leukemia, not otherwise classified AML, minimally differentiated AML, without maturation AML, with maturation Acute myelomonocytic leukemia Acute monoblastic or monocytic leukemia Acute erythroid leukemia Acute megakaryoblastic leukemia Acute basophilic leukemia Acute panmyelosis and myelofibrosis Myeloid sarcoma 14 of 46 Hegbom and Edenberg Treatment: 1st step is to obtain remission. Most patients are treated with a combination of anthracycline and cytarabine Acute promyelocytic leukemia (Fab M3) is treated with trans-retinoic acid and anthracylins. Once remission is entered, postremission therapy is given with curative intent. Options include standard chemotherapy and autologous/allogenic stem cell transplantation. Newer therapy: Gemtuzumab ozogamicin is a monoclonal antibody against CD33 (a molecule present on most AML cells but not on normal stem cells) conjugated to

calicheamicin (a potent chemotherapy molecule). Supportive care; blood products, antibiotics, growth factors. Prognosis: Cytogenetic analysis of the bone marrow is one of the most important prognostic factors. Patients with t(8;21), t(15;17) or inversion 16 have the best prognosis, with long-term survival rates of approximately 65%. Patients with normal cytogenetic findings have an intermediate prognosis and have a long-term survival rate of approximately 25%. Patients with poor-risk cytogenetic findings (especially -7, -5) have a poor prognosis, with a long-term survival rate of less than 10%. Increased age and antecedent hematologic disease give a poorer prognosis. 15 of 46 Hegbom and Edenberg 14. Myelodysplastic syndromes Myelodysplastic syndrome (MDS) refers to a heterogeneous group of closely related clonal hematopoietic disorders. All are characterized by a hypercellular or hypocellular marrow with impaired morphology and maturation (dysmyelopoiesis) and peripheral blood

cytopenias, resulting from ineffective blood cell production. All 3 cell lineages in myeloid hematopoiesis can be involved, including erythrocytic, granulocytic, and megakaryocytic cell lines. Although clonal, it is considered a premalignant condition in a subgroup of patients that often progresses to acute myeloid leukemia (AML) when additional genetic abnormalities are acquired. The disorders are usually idiopathic but may be seen after cytotoxic chemotherapy. As mentioned the MDS encompass several heterogeneous syndromes. Those with less than 5% bone marrow blasts are termed refractory anemia, with or without ringed sideroblasts. Those with excess blasts are diagnosed as refractory anemia with excess blasts (5-19% blasts). Those with a proliferative syndrome including peripheral blood monocytosis greater than 1000/µL are termed chronic myelomonocytic leukemia (CMML). Symptoms and signs: Most are over 60 years. Patients usually present with fatigue, infection, or bleeding related to

bone marrow failure. The course may be indolent, and the disease may present as a wasting illness Splenomegaly may be present (in CMML) Lab.: Anemia, usually macrocytic, and macro-ovalocytes may be seen on peripheral smear. Reticulocyte count is reduced The WBC count is usually reduced. Myeloid series may be left shifted, the platelet count is normal or reduced The bone marrow is usually hypercellular, but may be hypocellular. Aberrant cells Signs of abnormal erythropoiesis Ringed sideroblasts may be seen. Myeloid series is often left shifted (Separated form AML by presence of < 20% blasts) Common cytogenic abnormalities; loss or deletions of long arms of chr. 5 or 7 Treatment: RBC transfusions. Erythropoietin Severe neutropenia; G-CSF, GM-CSF Azacitidine improves both symptoms and blood counts and prolong time to conversion to AML. Other: Revimid (thalidomide analogue), antithymocyte globulin. Patients <60 years with matched sibling donors can be treated with allogenic bone

marrow transplantation. Prognosis: Ultimately fatal disease. Allogenic bone marrow transplantation is the only curative therapy The risk of transformation to AML depends on % of blasts in the bone marrow. Patients with refractory anemia may survive many years, and the risk of anemia is low. Those with excess blasts or CML have short survivals (< 2 years) and a higher risk of developing AML (20-50%). 16 of 46 Hegbom and Edenberg 15. ALL Side effects of chemotherapy Acute lymphoblastic leukemia is a clonal disease of the bone marrow in which there is a developmental arrest of early lymphoid precursors. These early lymphoid precursors proliferate and replace the normal hematopoietic cells of the marrow Consequently, anemia, thrombocytopenia, and neutropenia occur to varying degrees. The lymphoblasts also proliferate in organs other than the marrow, particularly the liver, spleen, and lymph nodes. ALL is the most common type of anemia in children and is more common in children

than in adults. The cause of ALL remains largely unknown, there are a few familial cases and a few cases associated with previous treatment with topoisomerase inhibitors. Signs and symptoms: Very similar to those of AML. See above Some patients may present with symptoms due to mediastinal masses (T-cell ALL), such as shortness of breath. Furthermore, leukostasis is much less common with ALL. Lab.: • • • • • • • A CBC with differential demonstrates anemia and thrombocytopenia to varying degrees. Patients with ALL can have a high, normal, or low WBC count, but usually exhibit neutropenia. Abnormalities in the prothrombin time/activated partial thromboplastin time/fibrinogen/fibrin degradation products may suggest concomitant DIC In the peripheral smear circulating blasts are usually seen, schistocytes may be seen if DIC is present. Serum LDH and uric acid are usually increased. Bone marrow: At least 20% blasts. A negative myeloperoxidase stain and a positive TdT is the

hallmark of the diagnosis of most cases of ALL. Samples should also be sent for cytogenetics and flow cytometry Cytogenetics -Approximately 15% of patients with ALL have a t(9;22) translocation (i.e, Philadelphia chromosome), but other chromosomal abnormalities also may occur, such as t(4;11), t(2;8), and t(8;14) Immunophenotying: Early precursor B-cell; TdT+, CD19+. Pre B-cell ALL; TdT+, CD19+, CD10+, cytoplasmic Ig+ B-cell; CD19+, CD10+/-, cytoplasmic Ig +/-, surface Ig +. Early T-cell precursor; TdT+, CD4/CD8 +/+ or -/- T-cell; TdT+, surface CD3+, CD4/CD8 +/- or -/+. Around 80% will have B-cell markers Treatment: ALL is treated initially with combination chemotherapy, including daunorubicin, vincristine, prednisone and aspirginase. As with AML, patients are treated with either chemotherapy or high-dose chemotherapy + bone marrow transplantation. Prognosis: The overall cure rate for childhood ALL is 80%. Older patients (and patients under age 2) fare less well Side effects of

chemotherapy: A number of cancer chemotherapeutic agents have cytotoxic effects on rapidly proliferating normal cells in bone marrow, mucosa, and skin. Still other drugs such as vinca alkaloids and taxanes produce neuropathy, and hormones often have psychological as well as physical effects. Appropriate dose modifications may minimize these side effects Dose modifications are usually considered in settings where treatment is given with palliative intent. Bone marrow toxicity – Autologous bone marrow or peripheral stem cell transplantation can reduce the myelosuppressive toxicity of chemotherapy. Growth factors that stimulate myeloid or erythroid proliferation are used too Chemotherapy-induced nausea and vomiting – Symptoms are thought to originate in CNS. 5-HT-3 antagonists are now the primary agents for treatment and prevention. These are more effective when given in conjunction with dexamethasone Other drugs used – Aprepitant, a substance P inhibitor. Metoclopramide, lorazepam

are often used in combination with other antiemetics Dronabinol is effective in some patients. One problem with all combinations is the development of tachyphylaxis GI and skin toxicity - Due to action on proliferating cells and immunosuppression. Miscellaneous – e.g Anthracyclines cardiomyopathy, interferon alfa; fever, weight loss, confusion A number of chemotherapeutic agents may cause secondary leukemia – procarbazine, melphalan, other alkylating agents, and etoposide. All alkylators can cause either temporary or permanent aspermia or amenorrhea. 17 of 46 Hegbom and Edenberg 16. Chronic myelologenous leukemia GAPA score Chronic myelogenous leukemia (CML) is a myeloproliferative disorder characterized by increased proliferation of the granulocytic cell line without the loss of their capacity to differentiate. Consequently, the peripheral blood cell profile shows an increased number of granulocytes and their immature precursors, including occasional blast cells. CML is an

acquired abnormality that involves the hematopoietic stem cell. It is characterized by a cytogenetic aberration consisting of a reciprocal translocation between the long arms of chromosomes 22 and 9; t(9;22). The translocation results in a shortened chromosome 22, termed the Philadelphia (Ph) chromosome This translocation relocates an oncogene called abl from the long arm of chromosome 9 to the long arm of chromosome 22 in the BCR region. The resulting BCR/ABL fusion gene encodes a protein with strong tyrosine kinase activity The presence of BCR/ABL rearrangement is the hallmark of CML, although this rearrangement has also been described in other diseases. It is considered diagnostic when present in a patient with clinical manifestations of CML. Approximately 5% of cases of CML are Ph chr negative, though molecular studies demonstrate BCT/ABL fusion gene. CML accounts for 20% of adult leukemias, it typically affects middle-aged adults. The disease consists of 3 phases: • Chronic

phase – Early CML, normal bone marrow function is retained, WBCs differentiate, neutrophils combat infection normally. However the disease is unstable and progress • Blast crisis - After an average of 3-5 years, the disease usually evolves into the blast crisis, which is marked by an increase in the bone marrow or peripheral blood blast count or by the development of soft tissue or skin leukemic infiltrates. Most blasts are of myeloid origin, in 1/3 of patients the blasts are of lymphoid origin. • Accelerated phase - In many patients, an accelerated phase occurs 3-6 months before the diagnosis of blast crisis. Symptoms and signs: The clinical manifestations of CML are insidious and are often discovered incidentally when an elevated WBC count is revealed by a routine blood count or when an enlarged spleen is revealed during a general physical examination. Nonspecific symptoms of tiredness, fatigue, and weight loss may be seen. Patients often have symptoms related to enlargement of

the spleen, liver, or both Some patients may have low-grade fever due to the hypermetabolic state. In blast crisis, patients may experience bleeding and infection related to bone marrow failure. Lab.: Hallmark of CML is an elevated WBC count; the median WBC count at diagnosis is 150000/µL. Peripheral blood; myeloid series is left shifted, mature forms dominating. At presentation the patient is usually not anemic Platelet count may be elevated Bone marrow; hypercellular, with left shifted myelopoiesis. Myeloblasts comprise less than 5% of marrow cells The leukocyte alkaline phosphatase score is low and is a sign of qualitative abnormalities in neutrophils. To calculate the GAPA score cells are stained for granulocyte alkaline phosphatase, subsequently 100 cells are examined and the degree of staining is scored from 14 in each cell (max. value 400) The reference range is between 40 and 140 The score in CML is usually below this The hallmark the disease is that the bcr/abl gene is

detected in the peripheral blood. In the accelerated and blast phases progressive anemia and thrombocytopenia occur. The % of blasts in the blood and bone marrow increases. Blast phase CML is diagnosed when blasts compromise more than 30% of bone marrow cells Treatment: Imatinib mesylate (Gleevec) results in nearly universal control of chronic phase disease. Development of resistance is a problem Longterm remissions are being evaluated Hydroxurea can be used for patients who do not tolerate imatinib. Interferon-α is also used Leukapharesis may be used if symptoms arise from hyperleukocytosis. The response to treatment is poorer in accelerated and blast phase. The only available potential curative therapy for CML is allogenic bone marrow transplantation. Best results is seen in bone marrow transplantation from HLA-matched siblings. Prognosis: With interferon therapy, median survival was 5-6 years. It is anticipated that imatinib will lead to improvements in survival rates. 18 of 46

Hegbom and Edenberg 17. Chronic lymphocytic leukemia CLL is a clonal malignancy of B-lymphocytes (98%). The disease is usually indolent, with slowly progressive accumulation of longlived small lymphocytes These cells are immunoincompetent CLL has a median age at presentation of 65 years. May be complicated by autoimmune hemolytic anemia or autoimmune thrombocytopenia. Symptoms and signs: Fatigue, lymphadenopathy. Enlarged spleen and liver may be seen With progressive disease there may be pallor due to anemia. Skin infiltration may be a feature of T-cell CLL Staging (useful prognostically); stage 0 – lymphocytosis only, stage I – plus lymphadenopathy, stage II - organomegaly, stage III – anemia, stage IV – thrombocytopenia. Lab.: Hallmark is lymphocytosis Usually 45-95% of the circulating cells are lymphocytes Lymphocytes are morphologically indistinguishable from normal small lymphocytes. Hct and platelet count are usually normal at presentation The bone marrow is variably

infiltrated with small lymphocytes. Immunophenotype demonstrates coexpression of CD19 with the T-cell marker CD5 Hypogammaglobuinemia is present in 50% of patients and becomes more common with advanced disease. Treatment: Most cases of early indolent CLL require no treatment. Indications for treatment include progressive fatigue, symptomatic lymphadenopathy, or anemia or thrombocytopenia. Fludarabine combined with rituximab is front–line therapy Prognosis: Stage 0-II, 5-20 years without treatment, stage III/IV, less than 2 years. With fludarabine combination therapy the 2 year survival is greater than 90%. 19 of 46 Hegbom and Edenberg 18. Polycythemia vera Essential thrombocytosis Polycythemia vera is an acquired myeloproliferative disorder that causes overproduction of all three hematopoietic cell lines, most markedly the red blood cells. Extramedullary hematopoiesis occur in the liver and spleen In primary polycythemia (polycythemia vera) the erythroid production is

independent of erythropoietin. Thrombosis, due to hyperviscosity, is the most common complication of polycythemia and the major cause of morbidity and death. Anemia, thrombocytopenia, and myelofibrosis may develop, and RBC and WBC precursors are released into the circulation. The median age at presentation is 60 years. Symptoms and sign: Most present with symptoms related to expanded blood volume and increased blood viscosity. Common complaints include headache, dizziness, tinnitus, blurred vision, and fatigue. Generalized pruritus may be a striking symptom and is related to histamine release from the increased number of basophils present. Bleedings (due to qualitative abnormalities in platelets) Physical examination reveals plethora and engorged retinal veins. The spleen is often palpable Signs and symptoms of thrombosis Lab.: Hct above normal The red blood cell mass is elevated RBC morphology is normal WBC count and platelet count is elevated The bone marrow is hypercellular, with

panhyperplasia of all hematopoietic elements. Hyperuricemia With myelofibrosis, anemia and thrombocytopenia may develop, with massive splenomegaly. Treatment: Treatment of choice is phlebotomy. One unit of blood is removed weekly until the hct is less than 45%, then maintained at this level by repeated phlebotomy. Aspirin is given to reduce risk of thrombosis Myelosuppresive therapy – may be used if response to phlebotomy is not satisfactory, and if there is intractable pruritis. Drug of choice is hydroxurea. Prognosis: Indolent disease with median survival of 11-15 years. May convert to myelofibrosis, CML, or AML Essential thrombocytosis is a clonal disorder of a multipotent hematopoietic stem cell. Marked proliferation of megakaryocytes in the bone marrow leads to elevation of the platelet count. Median age at presentation is 50-60 years Symptoms and signs: The most common symptoms are weakness, nonspecific headache, and paresthesias of the hands and feet. Signs and symptoms of

thrombosis. Bleeding is less common and is related to qualitative platelet defect Splenomegaly may be seen Lab.: Elevated platelet count The peripheral blood smear reveals large platelets The bleeding time is prolonged in some patients Treatment: Risk of thrombosis can be reduced by control of platelet count. Hydroxurea and anagrelide is used For vasomotor symptoms (parathesias, erythromelalgia) aspirin can be used. For immediate reduction in the platelet count, plateletpheresis has been used (e.g, in serious hemorrhage or thrombosis) 20 of 46 Hegbom and Edenberg 19. Bone-marrow transplantation The most important cell for transplantation is the hematopoietic stem cell. Currently the 3 major sources of stem cells for transplantation include the bone marrow, cord blood, and peripheral blood. When they are obtained from the recipient, they are called autologous. When they come from someone other than the recipient, they are termed allogeneic The 3 types of allogeneic donors are

syngeneic, related, and unrelated. When the donor is an identical twin, donation is termed syngeneic As the names imply, related allogeneic donors are relatives, and unrelated donors are identified through a donor registry or from a cord blood bank. Cellular Characteristics of Various Sources of Stem Cells Source Cellular Characteristics Bone Marrow Peripheral Blood Cord Blood Stem-cell content Adequate Good Low Progenitor-cell content Adequate High Low* T-cell content Low High Low, functionally immature Risk of tumor cell contamination High Low Not applicable * Studies have shown that the cord-blood progenitor cells have greater proliferative potential than that of peripheral blood and marrow progenitor cells Indications for transplantation: • Nonmalignant disorders; inherited metabolic disorders, inherited immune disorders, inherited red cell disorders, marrow failure states, autoimmune disorders (experimental). • Malignant disorders; ALL, AML, CML, juvenile

myelomonocytic leukemia, myelodysplastic syndromes, plasma cell disorders, Hodgkin and non-Hodgkin lymphoma The search process: For allogeneic transplants, human leukocyte antigen (HLA) histocompatability typing is performed for immediate family members initially using serologic typing. Fully matched family members provide the most compatible matches because they often share minor HLA antigens not usually included in testing. Class I and class II HLA antigen compatibility is tested and compared This information helps the transplant physician determine the risks of non-engraftment and GVHD. In addition, donor age (younger is better), sex (female stem cells given to a male is less favorable), cytomegalovirus (CMV) serology (CMV-negative has better outcome), pregnancy and transfusion history, and body weight are considered. The transplant process: The transplant process generally is divided into the following 5 phases: (1) conditioning, (2) stem cell infusion, (3) neutropenic phase, (4)

engraftment phase, and (5) postengraftment period. (1) The purpose is to deliver chemotherapy and/or radiation to eliminate malignancy, prevent rejection of new stem cells, and create space for the new cells. The most common conditioning regimens include total body irradiation (TBI) and cyclophosphamide or busulfan (Myleran, Busulflex) and cyclophosphamide. (2) Depletion of T cells can be performed to decrease GVHD. This often is performed before haplotype-matched transplants or other transplants that may have a significant degree of mismatch. Stem cell CD34+ selection may be performed, either for depletion of T cells or for tumor-purging purposes. The cells are infused through a central venous catheter, much like a blood transfusion (3) During this period (2-4 wk), the patient essentially has no effective immune system. Healing is poor, and the patient is susceptible to infection. Supportive care and empiric antibiotic therapy are the mainstays of successful passage through this phase

Fungal selection can occur. (4) During this period (several weeks), the healing process begins. The greatest challenges at this time are management of GVHD and prevention of viral infections (especially CMV). In hematopoietic cell transplantation the immune system is part of the transplanted organ; therefore, the new immune system can attack the entire body. When this occurs, it is termed GVHD This generally involves the skin, gastrointestinal tract, and the liver, causing rash/blistering, diarrhea, and hyperbilirubinemia, respectively. The good side of GVHD is the graft versus leukemic (GVL) effect that also may be present. (5) This period lasts for months to years. Hallmarks of this phase include the gradual development of tolerance, weaning off of immunosuppression, management of chronic GVHD, and documentation of immune reconstitution. Most patients need reimmunization, usually beginning 1 year posttransplant. Outcomes: Transplants for nonmalignant diseases generally have more

favorable outcomes, with survival rate of 70-90% if the donor is a matched sibling and 36-65% if the donor is unrelated. Transplants for acute leukemias (eg, ALL, AML) in remission at the time of transplant have survival rates of 55-68% if the donor is related and 26-50% if the donor is unrelated. Some words on autologous transplantation: Autologous transplantation refers to the use of the patients own stem cells as a rescue therapy after high-dose myeloablative therapy. This is generally used in chemosensitive hematopoietic and solid tumors to eliminate all malignant cells by administering high-dose chemotherapy with subsequent rescue of the hosts bone marrow with previously collected autologous stem cells. Immunosuppression is not required after autologous transplantation 21 of 46 Hegbom and Edenberg 20. Myelofibrosis (agnogen myeloid metaplasia and secondary myelofibrosis in myeloproliferative diseases) Bone marrow histology. Myelofibrosis: Primary myelofibrosis (agnogenic

myeloid metaplasia) - is a clonal disorder arising from the neoplastic transformation of early hematopoietic stem cells. AMM is categorized as a chronic myeloproliferative disorder The hematopoietic system is most affected Other organ systems may be involved by extramedullary hematopoiesis. The cause of the excessive marrow fibrosis observed in this disorder remains unclear. Platelets, megakaryocytes, and monocytes are thought to secrete several cytokines, such as transforming growth factor beta, platelet-derived growth factor, interleukin-1, epidermal growth factor, and basic fibroblast growth factor, which may result in fibroblast formation and extracellular matrix proliferation. The median age of diagnosis is 65 years. Symptoms and signs: Usually insidious in onset. Patients may present with fatigue, abdominal fullness due to hepatosplenomegaly Later symptoms of progressive bone marrow failure develop. Painful episodes of splenic infarction may occur Hematopoiesis in the liver leads

to portal hypertension. Lab.: Anemia, variable WBC count and platelet count Peripheral blood – the triad of teardrop poikilocytosis , leukoerythroblastic blood, and giant abnormal platelets is highly suggestive of myelofibrosis. The bone marrow can usually not be aspirated, though early in the disease fibrosis is detected by silver stain. Later, biopsy reveals severe fibrosis Treatment: No specific treatment. Anemic patients are supported with transfusion Androgens and erythropoietin may reduce transfusion requirement. Splenectomy indicated for splenic enlargement causing recurrent painful episodes, thrombocytopenia, or an unacceptable transfusion requirement. Allogenic bone marrow transplantation should be considered in younger patients Secondary myelofibrosis in myeloproliferative diseases – CML, AML, essential thrombocytosis. Bone marrow histology: See also topic 2. Normal architecture of red bone marrow; In sections, the hematopoietic cells appear to line in cords between

sinuses and between sinuses and bone. The cords of hematopoietic cells contain predominately developing blood cells and megakaryocytes. The cords also contain macrophages, mast cells and some adipose cells Specific types of blood cells develop in nests or clusters. Erythroblastic nests and megakaryocytes develop close to the sinuses, granulocytes farther away Bone marrow cellularity; hematopoietic content relative to the adipocyte content. Normally 40-60% of the non-bony tissue is composed of hematopoietic cells. However, cellularity varies with age and site (in elderly 20% cellularity may be normal) 22 of 46 Hegbom and Edenberg 21. Lymhogranulomatosis Hodgkin’s disease is a group of cancers characterized by Reed-Sternberg cells in an appropriate reactive cellular background. The RS cells represent a clonal proliferation of B lymphocytes that (T-cell derivation in 1-2%), in most instances, derive from the germinal centers of lymph nodes and that have lost their ability to

express their antibodies because of the introduction of multiple somatic mutations. EBV may be involved in the pathogenesis of Hodgkin’s disease There is a bimodal age distribution, with one peak in the 20s and a second over age 50, the disease is more common in males. An important feature of Hodgkin’s disease is its tendency to arise within single lymph node areas and spread in an orderly fashion to contiguous areas of lymph nodes. Only late in the disease there is vascular invasion Clinical findings: Most patients present because of a painless mass, commonly in the neck. Other symptoms are fever, weight loss, night sweats, or generalized pruritus. Subtypes: • Nodular sclerosis Hodgkin disease - 60-80% of all cases. • Mixed-cellularity Hodgkin disease - 15-30%. • Lymphocyte-depleted Hodgkin disease - Less than 1%. • Lymphocyte-rich classic Hodgkin disease - 5%. • Nodular lymphocyte-predominant Hodgkin disease - 5%. The typical RS cells are not observed, or appear

infrequently Staging: Stage I, one lymph node region involved; stage II, involvement of two lymph node regions on one side of the diaphragm; stage III, lymph nodes regions involved on both sides of the diaphragm; stage IV, disseminated disease with bone marrow or liver involvement. In addition, patients are designated stage A if they lack constitutional symptoms and stage B if 10% weight loss over 6 months, fever, or night sweats are present. Treatment: All should be treated with curative intent. Combination chemotherapy using doxorubicin (Adriamycin), bleomycin, vincristine, and dacarbazine (ABVD). Radiation therapy is used as initial treatment in stage IA and IIA disease Prognosis: Excellent prognosis for stage IA or IIA. Patients with disseminated disease (IIIB, IV) have 5 year survival rates of 50-60% 23 of 46 Hegbom and Edenberg 23. Non-Hodgkin lymphomas I – classification, diagnosis and differential These lymphomas compromise a group of heterogeneous cancers of lymphoid

origin, which have distinct features from Hodgkin lymphomas. Following the WHO guidelines, they can be divided into 4 main groups; precursor B cell, mature B cell, precursor T cell and mature T cell. Clinically they can be separated into indolent and aggressive The classification is based on cytogenetics, with each lymphoma having characteristics translocations, causing up regulation or down regulation of oncogenes or tumor suppressor genes respectively. Follicular lymphoma – t(14;18) Bcl-2/IgH Lymphoplasmacytic – t(9;14) PAX5 Mantle cell t(11;14) Bcl-1 Diffuse large B cell (t3;4) Bcl-6 Burkitt’s t(8;14)c-myc In terms of immunophenotype all B cells express CD20, while some express CD10 and CD43. T cells express CD3, 4, 8 and CD30 Standard investigations are full blood count, lymph node biopsy, bone marrow aspiration. Flow cytometry to classify the lymphoma Follicular lymphomas (mature B cell) comprise 20% of all NHL, and usually present as painless lymphadenopathy. Bone marrow

involvement is common, but the course of the disease is indolent with relapsing and remitting episodes. Progression to diffuse large B cell lymphoma carries poor prognosis. They are Cd5- , CD10+ and bcl-2+ Lymphoplasmacytic lymphoma is uncommon, but may present with severe bone marrow infiltration. Splenomegaly and anemia is frequent, and it may be associated with Waldenström’s macroglobulinemia. Symptomatic anemia or hyper viscosity is indicative of treatment. Large B cell lymphoma is the most common lymphoma, and is also fatal within months without therapy. Characterized by rapidly progressive lymphadenopathy. Burkitt’s lymphoma is endemic to west Africa, and is closely associated with Epstein-Barr virus infection. In addition to usual lymphoma findings, CNS involvement is common. Abdominal mass or jaw tumor are common presentations T cell lymphomas are much more rare than B cell lymphomas, although they do make up the majority of cutaneous lymphomas. Worth mentioning is primary

gastric lymphoma in association with H.pylori infection, and primary cutaneous lymphoma (sezary syndrome and mycosis fungoides). 24 of 46 Hegbom and Edenberg 24. Non-Hodgkin lymphomas II – therapy Treatment of lymphoma is like most cancers, divided into chemotherapy and radiotherapy, or combinations thereof. Side effects are many, and should be weighed against the overall prognosis. Read up on pharmacology Follicular lymphoma is, in the UK, treated with a COP regimen, consisting of cyclophosphamide, vincristine and prednisolone. Response is good, but complete remissions are rare. Other therapies are peripheral blood pool cell rescue (PBPCR) and addition of rituximab, which may increase chance of complete remission. Median survival is around 10 years Lymphoplasmacytic lymphoma follows supportive care in most cases. If associated with severe paraproteinemia, plasmapheresis may be an option. Chlorambucil has been the conventional treatment, but fludarabine has also been proven

effective Large B cell lymphoma is treated with CHOP+R, which consist of cyclophosphamide, hydroxydaunorubicin, vincristine, prednisolone and rituximab. Patients in stage I or II stand a good chance of cure after 3 to 6 cycles of this therapy If no response with CHOP+R is seen, PBPCR should be considered. Burkitt’s lymphoma is treated with cyclic combinations of cyclophosphamide, and methotrexate, as well as cytarabine in high doses. Complete remission rates are good, but hyperuricemia and tumor lysis syndrome may occur, and should be treated with rasburicase (recombinant urate oxidase). Of T cell lymphomas, primary gastric lymphoma is a surgical matter, and only treated with omeprazole for symptomatic relief. Primary cutaneous lymphoma should not be treated systemically, so either surgical or local irradiation. Of more experimental therapies, the newly emerged radioimmune therapy should be mentioned. In clinical trials, the 90Y conjugated CD20 monoclonal antibody ibritumomab and

the 131I conjugated CD20 tositumomab have shown promising results. 25 of 46 Hegbom and Edenberg 25. Follicular lymphomas MALT As previously mentioned, follicular lymphoma make up around 20% of all NHL B cell lymphomas. On presentation with lymphadenopathy, the diagnosis is established with fine needle aspiration and flow cytometry. The differential diagnosis of follicular lymphoma should include reactive hyperplasia, Hodgkin lymphoma, mantle cell lymphoma, marginal cell lymphoma and castleman’s disease. The flow cytometric profile of follicular lymphoma is that of CD5-, CD10+ and bcl-2 + cells. Various studies also indicate differences in intensity of CD19 and CD20 expression. Follicular lymphoma commonly have the t(14;18) translocation, but cases where this has been absent has been reported. In these cases there has been a translocation of bcl-6, which is known as a germinal center marker. Follicular lymphoma is graded histologically, in 3 grades. According to the WHO grade 1

is defined as less than 5 centroblasts/hpf, grade 2 is 5-15 centroblasts/hpf and grade 3 is more than 15 centroblasts/hpf. Grade 1 and 2 are considered low grade, while grade 3 is considered high grade. Treatment has been discussed. Mucosa-associated lymphoid tissue (MALT) lymphoma is mostly confined to the stomach and GI, but may be found in any glandular tissue in the body. It is usually found in the setting of a superimposed immune process in the respective tissue, such as sjögren’s before MALT lymphoma in the salivary gland and H.pylori infection before gastric MALT lymphoma It is considered a low grade follicular lymphoma, and treated as such. 26 of 46 Hegbom and Edenberg 26. Diffuse large B cell lymphoma Mantle cell lymphoma Diffuse large B cell lymphoma is a large cell lymphoma of B cell origin, showing diffuse growth. It exists in several subtypes, such as mediastinal, intravascular, lymphoid granulomatosis. It can arise from underlying follicular lymphoma It is

rapidly fatal without treatment, but with CHOP+R regimen 50% are now cured. Mantle cell lymphoma, previously centrocytic lymphoma, is a small cell lymphoma. It comprises 6% of all NHL and has a median survival of 3-4 years. Histologically it is characterized by expanding mantle zones, surrounding compressed germinal centers Histological subtypes are diffuse, nodular, mantle zone and blastic. The sine qua non in the diagnosis is the bcl-1 expression It is an aggressive lymphoma resistant to chemotherapy, but CHOP+R treatment should be attempted. Also being investigated are bortezomib and genasense, a proteasome inhibitor and a bcl-2 blocker respectively. 27 of 46 Hegbom and Edenberg 27. Multiple myeloma and Waldenström’s macroglobulinemia Multiple myeloma is a tumor of the plasma cells in the bone marrow. The presenting complaint is usually bone pain, as the myeloma cells induce expression of RANK ligand in the stromal cells, promoting osteclastics activity. Furthermore,

recurrent infections, anemia and hypercalcemia might also be seen. It usually affect people over 60 years. The main diagnostic finding is paraproteinemia, which usually is IgG, IgA or IgD in that order. Bone marrow smear will show diffuse infiltrate of plasma cells. Lytic bone lesions is easiest seen on skull x-ray, and helps differentiate myeloma from monoclonal gammapathy of unknown significance (MGUS) and Waldenström’s. The currently most effective treatment is a combination of thalidomide and dexamethasone, which has been shown to be superior to the vincristine-doxorubicin-dexamethasone regimen. A combination of lenalidomide, a thalidomide derivative, and bortezomib appears to be effective. Concomitant hypercalcaemia should be controlled with zoledronate Median survival is around 5-6 years, and the serum level of β2-microglobulin along with albumin levels is the new staging system. The previous staging, the Salmon-Durie system, was based on paraprotein levels, blood coutn and

number of lytic lesions. Waldenström’s macroglobulinemia present with non-specific symptoms, but splenomegaly may be present. It is a malignancy of B cells, that are believed to be a hybrid of lymphocytes and plasma cells. Patients are usually elderly, with fatigue related to anemia as the main complaint. In diagnostics, the blood smear will show roleaux formation of the blood cells. Serum electrophoresis will show a monoclonal IgM spike, which distinguishes it from multiple myeloma. It differs from MGUS by bone marrow infiltration by plasmacytic lymphocytes In cases with severe hyperviscosity syndrome, with stupor or coma, emergency plasmapheresis is the treatment of choice. Autologous stem cell transplant is an option, but mainly considered in young patients with aggressive disease. Median survival is 3-5 years 28 of 46 Hegbom and Edenberg 28. Normal hemostasis Current concept Hemostasis is the maintenance of hemodynamic stability, tightly controlled by the balance between

procoagulant factors and anticoagulant factors. In essence its 2 overlapping pathways, the platelets and the fibrinogenesis Platelets adheres to subendothelial structures trough glycoprotein Ia and glycoprotein Ib, and the latter does so with activated Factor VII in between. Following platelet adhesion, they undergo a change from discoid shape to spherical, and release their dense bodies and α-granules. The dense bodies contain ADP and serotonin, and the –granules contain PDGF, platelet factor 4(heparin antagonist), fibrinogen, thrombospondin. The released ADP activates fibrinogen receptor and glycoprotein IIa-IIIb receptor, which both mediate platelet aggregation. In fibrinogenesis, which forms the concrete between the platelets, we differentiate between the extrinsic pathway and the intrinsic pathway. Common to both of them is a cascade of serine proteases activating the next step in the sequence Here the intrinsic pathway seen starting with factor XI, and the extrinsic with TF +

VIIa. It follows from the above flow chart that the tissue factor – factor VII complex activates factor X, which in turn cleaves prothrombin to thrombin. Thrombin, in turn binds to thrombomodulin on the platelet surface and activates protein C, which combines with protein S to inactivate factors VIIIa and Va, to oppose coagulation. Protein C also inactivates plasminogen activator inhibitor 1, which controls activation of tissue- plasminogen activator. The t-PA converts plasminogen to plasmin, which in turn convert fibrin to FDPs and Ddimer Bleeding time is measured by 1mm deep 1cm long incisions on the arm, with cuff applied, and should be 3-10 min. Prothrombin time measures the extrinsic pathway, and should be 16-18 seconds. Activated partial thromboplastin time measures intrinsic pathway, and should be 30-50 seconds. Thrombin time is prolonged in fibrinogen deficiency or dysfibrinogenemia, normal value is around 12 seconds. Remember that platelet disorder present with spontaneous

bleeding of smaller vessels, with petechia and purpura. Mucous membrane bleeding is also common. In coagulation disorders, hemarthroses or muscle hematoma is common, often preceded by an initial event such as a minor injury. 29 of 46 Hegbom and Edenberg 29. Vascular bleedings Vascular bleedings are characterized by easy bruising and bleeding into the skin. The pathophysiology lies within the vessels themselves, and can be divided into congenital and acquired. Congenital are hereditary hemorrhagic telangiectasia, and the connective tissue disorders such as Ehlers-Danlos, pseudoxanthoma elasticum and marfan’s syndrome. In hereditary hemorrhagic telangiectasia, and autosomal dominant disorder caused by deletion in ENG or AVCRL1, the cause of bleeding is rupture of arteriovenous malformations. Epistaxis at night is a common finding Definitive diagnosis is with qPCR or southern blot. Treatment is avoiding injury, and possibly surgical embolization of larger AVMs in different

organs. In acquired causes we can divide into severe infection, allergic (henoch-schönlein purpura), drugs ( steroids, sulphonamides). Treatment is aimed at the underlying cause. 30 of 46 Hegbom and Edenberg 30. Thrombocytopenias Thrombocytopenias are defined as a reduction in platelet numbers below 150 T/l, although clinical manifestations may not occur before <50 T/l, and then in the form of injury induced hemorrhage or purpura. As with anemia, thrombocytopenia can be grossly divided into impaired production or increased destruction. Impaired production follows all types of bone marrow failure, myeloma, leukemia, vitamin deficiency or acquired aplastic anemia. Increased destruction can be immune, hypersplenism, dilution, DIC or TTP Idiopathic thrombocytopenic purpura is an antibody mediated destruction of platelets in the spleen. In adults it is commonly seen in association with SLE, thyroid disease or AIHA. Auto antibodies typically target gp IIa/IIIb receptors Easy

bruising, epistaxis and menorrhagia is common. First line treatment is high dose prednisone Intravenous IgG is effective, but reserved for emergency bleedings. Splenectomy is second line treatment In patients who don’t respond to splenectomy either, danazol can be attempted Heparin induced thrombocytopenia is a rare complication of heparin therapy, but can occur 5-14 days after initiated therapy. HIT is associated with severe thrombosis, and heparin must be discontinued immediately, and coagulation maintained by hirudin, danaparoid or ximelagatran. Thrombotic thrombocytopenic purpura presents with purpura, fever, neurologic deficits and hemolytic anemia. The thrombocytopenia is thought to arise from increased multimers of factor VII, which under normal circumstances are cleaved by the gene product of ADAMTS13. Normal coagulation tests help differentiate it from DIC Emergency treatment is plasmapheresis, to reduce numbers of fVII multimers, and provide the protease. Hemolytic uremic

syndrome is believed to represent the same entity as TTP, with predominatly renal failure and less neurologic deficits. In children it frequently is associated with infection by salmonella, shigella or Ecoli strain O157:H7 Peripheral blood smear will show fragmented RBCs, due to microangiopathic hemolytic anemia. The prognosis is closely related to renal function, but treatment options are the same as for TTP. 31 of 46 Hegbom and Edenberg 31. Platelet disorders Disorders affecting platelet function can be divided into acquired and inherited. Of the more important inherited are Glanzmann’s thrombastenia, Bernhard-Souliers and Storage pool disease. In Glanzmann’s, which is an autosomal recessive disorder, is a deficiency of gp IIa/IIIb receptors. Platelet numbers are normal but bleeding time is increased. In Bernhard-Soulier there is a deficiency of the glycoprotein Ib-V-IX complex In storage pool disease there is defective secretion of platelet granules. In these diseases

platelet transfusion is an option, but some may respond to DDAVP Of acquired disorders uremia causes abnormal platelet function by uknown mechanisms. May respond to DDAVP Myeloproliferative can also cause abnormal platelet function, while increasing platelet numbers, in these cases plasmapheresis might be an option. Aspirin is obviously also a cause of platelet dysfunction. 32 of 46 Hegbom and Edenberg 32. von Willebrand disease In von Willebrand disease, there is a defective platelet function as well as a deficiency in factor VIII-C complex, both as a result of vWF dysfunction. By mechanism, 3 subtypes can be identified In type 1 there is a quantitative decrease in vWF, and this accounts for 80% of the cases. In type IIa, there is dysfunction of the protein that prevents multimer formation In type IIb there is a dysfunction in the protein that mediates clearance of the multimers. In type III vWF is nearly absent Pseudo-von willebrand disease is a rare disorder where the

platelet membranes have increased avidity for the large vWF multimers. Symptoms are characteristic of platelet dysfunction, with epistaxis and menorrhagia. Aspirin use may reveal the disease Assay of facto VIII-C complex will distinguish vWF disease from all others except hemophilia. Hemophilia is ruled out by bleeding time and/or level of factor VIII antigen. Except type IIb, they may all respond to DDAVP The factor VIII concentrate feiba may also have a role in treatment. 33 of 46 Hegbom and Edenberg 33. Hemophilia A and B Hemophilia A is an x-linked recessive disorder which primarily affects males. It is characterized by low factor VIII-C activity, and spontaneous hemarthroses is the hallmark of the diagnosis. Bleeding time and coagulation parameters are normal Clinically Hemophilia A cannot be distinguished from Hemophilia B, and specific assays are needed. Treatment range from DDAVP in mild cases, to factor VIII concentrates and the fibrinolytic agent ε-amino-caproic acid.

In the case were Hemophilia A is acquired, the problem is auto antibodies against factor VIII. This should be suspected in any bleeding disorder with prolonged PTT. Treatments include prednisone, feiba and novoseven Hemophilia B is also x-linked recessive, with low levels of factor IX coagulant. It presents clinically the same as hemophilia A, with prolonged PTT. Treatment of choice is factor IX concentrates 34 of 46 Hegbom and Edenberg 34. Other factor deficiencies and acquired coagulopathies Factor XI deficiency is an autosomal recessive disorder primarily seen among jews. PTT is prolonged and specific assay is needed to establish the diagnosis. Treatment is fresh frozen plasma when necessary Isolated deficiencies of factor II, V, X or VII are extremely rare. In case of factor XIII deficiency, may cause delayed bleeding time, but normal coagulation tests. Diagnosis is made by clot stability in 8M urea Afibrinogenemia is a rare disorder in which fibrinogen is absent. PT and

PTT are markedly prolonged Patients may have symptoms resembling severe hemophilia. Treatment is fibrinogen replacement by cryoprecipitate Coagulopathy of liver disease is the diminishing clotting factor levels due to longstanding liver failure. Liver disease first affects the vitamin K dependent factors; factor II, VII, IX and X. In addition, increased fibrinolysis also occurs due to α-antiplasmin Malabsorption of vitamin K due to biliary disease is another mechanism by which liver disease may cause bleeding tendencies. Only treatment of choice in liver related coagulopathy, is fresh frozen plasma. 35 of 46 Hegbom and Edenberg 35. Approach to the bleeding patient: bleeding types (vascular, platelet, coagulation) Vascular bleedings are characterized by easy bruising and bleeding into the skin. The pathophysiology lies within the vessels themselves, and can be divided into congenital and acquired. Congenital are hereditary hemorrhagic telangiectasia, and the connective tissue

disorders such as Ehlers-Danlos, pseudoxanthoma elasticum and marfan’s syndrome. In hereditary hemorrhagic telangiectasia, and autosomal dominant disorder caused by deletion in ENG or AVCRL1, the cause of bleeding is rupture of arteriovenous malformations. Epistaxis at night is a common finding Definitive diagnosis is with qPCR or southern blot. Treatment is avoiding injury, and possibly surgical embolization of larger AVMs in different organs. In acquired causes we can divide into severe infection, allergic (henoch-schönlein purpura), drugs ( steroids, sulphonamides). Treatment is aimed at the underlying cause. Platelet bleedings can be defined as either a quantitative or qualitative disorder of the platelets. In either case, coagulation parameters are normal, with increased bleeding time. Excessive bruising and bleeding time are characteristic Mucosal bleedings are common, and bleeding after dental procedures may be a first presentation. Bleedings are uncommon with more than 50

G/l, while severe bleedings are common with count of less than 20 G7/l. Platelet bleedings can, in general, be treated with fresh frozen plasma and in many cases DDAVP. Coagulation disorders can be inherited or acquired. In the case of hemophiliacs, the hallmark is spontaneous hemarthroses Increased bleeding tendency is a hallmark of coagulation disorders. Factor concentrates are the mainline treatment in addition to fresh frozen plasma. 36 of 46 Hegbom and Edenberg 36. Hereditary thrombophilia Thrombophilia are by definition a predisposition to arterial or venous thrombosis. Clinically, recurrent thrombotic events, or first incident in young age should lead to suspicion of thrombophilia. Of hereditary thrombophilias the most important are, Factor V Leiden, prothrombin variant, antithrombin deficiency and protein C/S deficiency. Factor V Leiden is caused by a SNP in the factor V gene. This mutant factor V is resistant to cleavage by protein C, and thus predisposes to

thrombosis. It is found in 3-5% of healthy individuals in the western world Contraceptive use in patients with factor V Leiden will significantly increase the risk for thrombosis. Prothrombin variant is due to a SNP in the 3’-UTR in the prothrombin gene. This causes a rise in the prothrombin levels and a threefold increase in the risk of venous thrombosis. Antithrombin deficiency is an autosomal dominant disorder associated with decreased levels of antithrombin. These patients are naturally resistant to heparin, and must be anticoagulated with hirudin, fondaparinux or ximelagatran. Protein C/S deficiencies are autosomal dominant disorders. Homozygous variants cause neonatal purpura fulminans, which is fatal and require immediate replacement therapy. Dysfibrinogenemia is malfunctioning assembly of the fibrin cross links, and is characterized by prolonged reptilase time. Prolonged reptilase time is also seen In liver coagulopathy. 37 of 46 Hegbom and Edenberg 37. Aquired

thrombophilia In the setting of acquired prothrombotic states, 2 main etiologies should be considered, malignancy and medications. Some rarer forms, such as paroxysmal nocturnal hemoglobinuria and ulcerative colitis-induced, fall outside this setting. Naturally, septicemia can cause DIC, but it would be farfetched to call an infection a source of acquired thrombophila because of it. Lupus anticoagulant is associated with SLE. Although PTT and bleeding time may be prolonged, there is increased risk for thrombosis The Russell viper venom time is a sensitive assay which specifically demonstrates the presence of lupus anticoagulant. Whenever a markedly prolonged PTT is seen without bleeding, lupus anticoagulant should be suspected. Prednisone efficiently reduces antibody concentrations. Paraneoplastic thrombophila has unknown pathomechanism, but the existence of a cancer procoagulant has been hypothesized. This is believed to be a cysteine protease causing activation of factor X. It can

occur as both arterial and venous thrombosis Venous thrombosis associated with cancer may occur in unusual places, such as mesenteric or splenic vein. As for medications, estrogen stands out as the most important causative agent. Estrogens have an effect on both lipid and carbohydrate metabolism, and the thrombophilia is believed to be an indirect cause of this. Heparin-induced thrombocytopenia is a rare, but important cause of sudden worsening in patients receiving heparin. It can also happen with LMW heparin, but the incidence appears to be lower. The theory behind transient protein C depletion upon starting anticoagulation therapy with warfarin, only has clinical value in patients with undiagnosed protein C deficiency. In these cases capillary thrombosis and skin necrosis may be seen 38 of 46 Hegbom and Edenberg 38. DIC Disseminated intravascular coagulation is associated with any number of serious underlying diseases, such as septicemia or AML. It is defined as a consumption

coagulopathy, due to the overactivation of the clotting cascade in the microcirculation. What triggers this event is uncertain, but an overexpression of tissue factor on monocytes and endothelial cells may be the inciting event. The overactivation and consumption of coagulation factors is followed by fibrinolysis, which is why DIC is just as often associated with bleeding as thrombosis. Secondarily, it may also produce microangiopathic hemolytic anemia Bleeding may occur anywhere, but spontaneous bleeding at venipuncture sites should be indicative. Subacute DIC is seen in cancer patients with superficial thrombosis, known as Trousseau’s syndrome. Treatment is mainly supportive. Clinical trials have been performed both with recombinant antithrombin and recombinant activated protein C. Only rAPC where shown to improve 28-day survival, and drotrecogin alpha (xigris) is currently the only agent available Antithrombin III levels could be monitored to establish if the DIC will cause

bleeding or thrombosis, but the clinical value of this is uncertain. 39 of 46 Hegbom and Edenberg 39. TTP/HUS Thrombotic thrombocytopenic purpura is an uncommon disorder with microangiopathic hemolytic anemia, thrombocytopenia and elevated serum LDH. It is commonly seen in young adults, with a slight female predominance It has been associated with use of ticlopidine and quinine. The cause has been linked to deficiency of ADAMTS13, a factor VII cleaving protein. Absence of this protein causes large multimers of factor VII which agglutinate and adhere to the endothelium. Anemia is present, and there may be marked reticulocytosis. Schistocytes can be seen on peripheral blood smear Thrombocytopenia is always present. Coagulation tests are normal, unless TTP is complicated by DIC. In differential diagnosis, other causes of microangiopathic hemolytic anemia should be excluded. DIC is excluded by normal coagulation parameters Evan’s syndrome (comb of autoimmune thrombocytopenia and

autoimmune hemolytic anemia) will show spherocytes in peripheral blood smear, and not schistocytes. TTP cannot easily be distinguished from HUS, the 2 diseases should be considered as a spectrum, where TTP has predominantly neurologic deficits, and HUS predominantly has renal failure. The treatment is large-volume plasmapheresis, with fresh frozen plasma replacement. Hemolytic uremic syndrome is believed to have the same pathomechanism has TTP, but occurs more frequently in children and in the setting following infection of shigella, salmonella and E.coli O157:H7 Presenting symptoms may be anemia, bleeding or renal failure. In children, HUS is usually self-limited and requires no further treatment. For adults, the treatment is the same as for TTP 40 of 46 Hegbom and Edenberg 40. ITP Idiopathic thrombocytopenic purpura is an autoimmune disorder with IgG formed against platelets. The platelets are then destroyed by platelets in the spleen. It commonly occurs in childhood, and

presents with mucosal or skin bleeding Epistaxis, oral bleeding or menorrhagia along with splenomegaly should lead to suspicion. Severe thrombocytopenia is present and coagulation parameters normal. Bone marrow examination is necessary to rule out myelodysplasia, although isolated thrombocytopenia is nearly synonymous with ITP. Initial treatment is prednisone, and 80% will respond to this within 3 weeks. However, recurrent thrombocytopenia will occur in most patients if prednisone is withdrawn. An alternative is dexamethasone Final treatment is splenectomy, which give complete remissions in some cases. Intravenous immunoglobulin is effective in raising platelet count fast, but due to price, only suitable for ITP induced bleeding emergencies. 41 of 46 Hegbom and Edenberg 41. Treatment and care of venous thromboembolic patients Venous thrombosis may in theory occur in any part of the venous circulation, but the vast majority seen in clinical practice involves venous thrombosis of

the leg. In addition to the already discussed causes of acquired and inherited thrombophilia, prolonged bedrest itself can predispose to venous thrombus formation. In isolated cases of venous thrombosis, with no other risk factors present, malignancy should always be suspected. On presentation, the leg may have edema, erythema and pain. In terms of physical examination, Homan’s and Löwenbrau give some indication. Blood test for d-dimer and FDP further support the diagnosis Definitive diagnosis is with compression ultrasonography Invasive venography has been abandoned by most, due to its complications and lack of additional clinically relevant information. Venous thrombosis in the upper extremity may present as unilateral swelling, erythema, pain and superior vena cava syndrome. In neonatology, thrombocytopenia may be the only evidence of venous thrombosis. The one and only feared complication of venous thrombosis is pulmonary emboli, and the majority of patients presenting with

venous thrombosis have had symptomatic or asymptomatic pulmonary emboli. The mainstay treatment of venous thrombosis is anticoagulation. This is usually achieved by enoxaparin the first 48 hours, before switching to syncumar. The target INR in venous thrombosis is 2-3, and up 25-3-5 in high risk patients Enoxaparin can be substituted with fondaparinux, which has so far not caused any incident of HIT. Syncumar was thought to be replaced by ximelagatran, a direct thrombin inhibitor, which unlike syncumar, requires no monitoring. However, it was withdrawn due to hepatotoxicity Surgically, an inferior vena cava filter has been shown to prolong survival in patients with lower extremity venous thrombosis, but this is currently only considered for patients who are unable to take anticoagulation therapy. 42 of 46 Hegbom and Edenberg 42. Anticoagulant therapy, prophylaxis The main goal in anticoagulation is prevention of clot formation. This is today mainly achieved with heparin, either

as unfractionated heparin or low molecular weight heparin. Whereas unfractionated heparin has an equal effect on both thrombin and factor Xa, low molecular weight heparin acts more on factor Xa. The benefits of low molecular weight heparin over unfractionated heparin are no need for APTT monitoring and less incidence of thrombocytopenia. The vitamin K antagonists (warfarin, coumarin, syncumar) have the advantage of oral administration. They are however, less effective than LMW heparin in preventing venous thrombosis, and requires complicated dosing regimens to maintain a stable INR. Fondaparinux is a synthesized pentasaccharide, which only has activity against factor Xa. This has the advantage of no activity against platelets, and longer half-life. Danaparoid is a heparinoid, meaning that it has activity against factor Xa, but is chemically distinct from heparin so it may be better tolerated in cases of HIT. The direct thrombin inhibitors in clinical use are argatroban, lepirudin,

bivalirudin and hirudin. They are mostly indicated in cases of ATIII deficiency or in HIT. Other anticoagulants are defibrotide, which increases PGE2 production and thus alters platelet activity. It is mostly indicated in peripheral arterial disease. The anticoagulant effect is mild compared to LMW heparins Dermatan sulfate has been investigated as an anticoagulant. It inactivates thrombin through heparin cofactor II, thus bypassing antithrombin III. It has been shown to be more effective than heparin in some studies, and may be used in HIT 43 of 46 Hegbom and Edenberg 43. Arterial thrombosis Arterial thrombosis encompasses everything from stroke, myocardial infarction to peripheral vascular disease. It is the leading cause of death in the western world. The vast majority of arterial thrombosis arises from arteriosclerosis A less significant fraction may be caused by paradoxic emboli or malignancy. The symptoms and treatment of AMI and stroke is beyond the scope of this review.

The manifestations of peripheral vascular disease however, may be distinct from venous thrombosis. Whereas a venous occlusion in the leg will cause swelling, erythema and pain, an arterial occlusion will cause cold and white extremities. Hence the term, phlegmasia alba dolens (white pain) which is used about femoral artery occlusions. Intermittent claudication refers to the pain the patient experiences when using muscles in the underperfused area. The distance walked before this pain manifests, is used as a marker of progression of the disease Claudication is however just a sign of underperfusion, usually in the calf, and not of thrombosis per se. The differential diagnosis could include buerger’s disease, trauma or popliteal entrapment syndrome. The anamnesis should however easily exclude these. In case of arterial thrombosis, the definitive diagnosis is also here ultrasonography. In case of thrombosis of larger vessels, such as the femoral artery, immediate thrombolysis is

indicated. Either intravenous rt-PA (tenecteplase, reteplase) Intraarterial injection of urokinase or defibrase may be superior, but requires competence at site. In case of popliteal artery thrombosis, percutanous transluminal angioplasty can be achieved. In terms of peripheral vascular disease in general, angiogenesis is the therapeutic goal. The methods used in attempts to achieve this are currently trental , pletal and praxilene. Autologous hematopoietic stem cell transplant has also been attempted The clinical effects of these are however, questionable. 44 of 46 Hegbom and Edenberg 44. Laboratory methods in the disorders of hemostasis The most common methods used to detect abnormalities in hemostasis are aPTT, PT, TCT, bleeding time and d-dimer. Activated partial thromboplastin time monitors the intrinsic pathway, and the normal value is 25- 39 seconds, although this varies between laboratories. An increase in aPTT may indicate heparin use, lupus anticoagulant or hemophilia

Prothrombin time, and consequently INR, measures the extrinsic pathway, more specifically factors II, V, VII, X and fibrinogen. The normal value is around 12-15 seconds. Prolonged PT may indicate warfarin usage, liver coagulopathy, DIC, vitamin K deficiency or factor VII deficiency. The international normalized ratio is calculated from the international sensitivity index, and should be between 0.8 and 12 Thrombin clotting time, or thrombin time, is used to monitor heparin therapy and fibrinogen levels. Normal range is less than 21 seconds, and values higher than this may indicate heparin use, lupus anticoagulant or hypofibrinogenemia. Bleeding time is determined by a standardized cut on the patients forearm, and should be 2-9 minutes. This is highly platelet depending, so a prolonged bleeding time indicates either decreased platelet number or intrinsic platelet dysfunction. Suspect thrombocytopenia, von Willebrand disease, DIC or aspirin use. D-dimer indicates ongoing fibrinolysis,

and a positive test is indicative of venous thrombosis, pulmonary emboli or DIC. Other tests include specific assays for factors. The diluted russel viper venome test is used for lupus anticoagulant Ristocetin cofactor test is used for von willebrand disease. Thromboelastography is a test of the efficiency of coagulation, and incorporates both plasma coagulation and platelet function. 45 of 46 Hegbom and Edenberg 45. Antiplatelet drugs An antiplatelet drug simply blocks platelet function in one way or another, thus inhibiting either activation or aggregation. The by far most used antiplatelet drug is aspirin, which irreversibly inhibits COX-1 by acylation, thus blocking synthesis of TXA2 and activation of platelets. Further evidence suggest a modification of COX-2 towards lipoxins, which may have anti-inflammatory effects Recent data also suggest a role in modulation of NFκB. The ADP receptor antagonists clopidogrel and ticlopidine block the ADP receptor P2Y12 which stops the

gp IIb/IIIa pathway to aggreagation. Clopidogrel has less side effects than ticlopidine, but they have both been associated with neutropenia and TTP The only phosphodiesterase inhibitor in use is cilostazol, which selectively blocks the cAMP pathway involved in platelet activation. It also causes arteriolar vasodilation, and is mainly used in peripheral vascular disease. The glycoprotein IIb/IIIa receptor antagonists are reopro, integrillin and aggrastat. These drugs directly block the cross-linking of platelets. They are very potent, and severe hemorrhage is a real concern Dipyridamole is an adenosine reuptake inhibitor, which blocks uptake of ADP in red blood cells and endothelial cells. Due to its additional vasodilating effect, it is also used in myocardial stress testing. 46 of 46