PATHOLOGY OF THE HEMATOLYMPHOID SYSTEM 1 AND 2 -INTRODUCTION TO ANEMIA - ANEMIA OF DECREASED PRODUCTION.
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1 PATHOLOGY OF THE HEMATOLYMPHOID SYSTEM 1 AND 2 -INTRODUCTION TO ANEMIA - ANEMIA OF DECREASED PRODUCTION. 1
2 ANEMIA Anemia is defined as decrease in the RBC mass, it usually is reflected by decrease in hemoglobin and hematocrit levels, usually not always as we shall see below. The reason hemoglobin and hematocrit are used instead of RBC mass is that he latter is a difficult and expensive assay to perform; it is used mainly in large centers for research purposes. To understand anemia, it is vital to be familiar with the various parameters that constitute the complete blood count (CBC) RBC count: the number of RBCs per unit volume, usually expressed in number (usually in millions) /microliter, for example 5x10 6 /microliter (μl) Hemoglobin concentration (Hgb): the amount of hemoglobin present in a unit volume, typically expressed in gm/dl Hematocrit (Hct): the ratio of the length of the tube occupied by cells over the whole length of the tube, expressed in percentage points. Mean cell volume (MCV): the average volume of a red cell expressed in femtoliters (fl=10-15 liter) Mean cell hemoglobin (MCH): the average content (mass) of hemoglobin per red cell, expressed in pictograms (pg=10-12 g) Mean cell hemoglobin concentration (MCHC): the average concentration of hemoglobin in a given volume of packed red cells, expressed in grams per deciliter. Red cell distribution width (RDW): the coefficient of variation of red cell volume, expressed in percentage points. Reticulocyte count: reticulocytes are immature, anucleated RBCs, expressed as percentage or as number/volume. 2
3 Here are some pearls regarding the above mentioned parameters RDW is useful in differentiating iron deficiency anemia from thalassemia (both have low MCV), RDW is high in iron deficiency anemia and normal to low in thalassemia MCV is the basis of classification of anemias into microcytic, normocytic and macrocytic Spherocytosis is the only anemia with high MCHC Reference ranges vary from place to place, and it is vital that you look at the reference range provided by the lab that performed the test rather than memorizing them. Reticulocyte count helps distinguish anemia resulting from decrease production vs anemia of increased peripheral destruction. 3
4 This is a schematic depiction of how hematocrit is measured, basically an anticoagulated specimen is centrifuged and the length occupied by the cellular component (packed RBCs, platelets and WBCs) is measured and divided by the whole length of the specimen. Four of the parameters the describe RBCs are measured (RBC count, RDW, hemoglobin concentration and hematocrit); the other three parameters can be calculated as follows, - MCH= Hgb/RBC count - MCV= Hct/ RBC count - MCHC= Hbg/Hct 4
5 CBC is performed in a test tube that has EDTA additive (tube with a lavender top) in it to prevent coagulation. As mentioned previously, RBC mass is a difficult assay and Hct and Hgb are used clinically to assess anemia, however there some scenarios in which the patient is anemic with normal Hct and Hgb and vice versa - Patients with acute blood loss, such as traumatic hemorrhage in a car accident, loses equal amounts of plasma and packed RBCs, hence, the patient will have normal CBC at the time of presentation, but in reality the patient is anemic as he has lost a significant amount of RBCs. - On the other hand a patient with fluid overload, during pregnancy for example as pregnant women retain extra amounts of fluid, will have a decreased Hct and Hgb, but are not considered anemic since no RBC loss was sustained. 5
6 CLINICAL MANIFESTATIONS OF ANEMIA The role of RBCs is to deliver oxygen to tissue in the right amount for the basic functions to meet the demands, a decrease in the RBC and Hgb means decreased capacity to deliver oxygen, the body will try to compensate for the deficit and in that case no symptoms will develop, however, if the compensatory mechanisms are exceeded by the anemia, symptoms will appear: - Symptoms related to decrease Hgb concentration include pale skin and pale conjunctiva - Symptoms related to decrease oxygen delivery include fatigue, dizziness, faintness, chest pain (and myocardial infarction in severe cases), and muscle weakness - Symptoms related to compensatory mechanisms by the body trying to overcome the anemia include shortness of breath (tachypnea) and tachycardia. Additionally, in some forms of anemia, splenomegaly (enlarged spleen) might develop as synthesis of blood shifts from the bone marrow to the spleen. - Symptoms related to the cause of anemia; menorrhagia, weight loss in cases of colon cancer, symptoms related to kidney failure, manifestations of chronic rheumatologic disorders etc. PLEASE REMEMBER - The development of symptoms are related to the underlying status of the patients and their physiologic reserve; symptoms will be felt more readily by a seasoned athlete suffering a small degree of anemia than a patient who lives a sedentary lifestyle. Additionally, a young healthy patient will tolerate a larger degree of anemia compared to an elderly patient with underlying cardiac or respiratory diseases. - The onset of anemia also plays a role in the development of anemia- related symptoms, for example iron deficiency anemia might be tolerated and completely asymptomatic if occurs over an extended period of time compared to anemia happening acutely as a result of massive blood loss, for example. WORK UP In addition to taking good history that includes the patient symptoms, past history, medications and other chronic diseases, thorough physical examination, and a panel of tests might be employed to further classify and diagnose anemia, and hence dictate the proper course of treatment; these include: 1- Iron indices (serum iron, serum iron- binding capacity, transferrin saturation, and serum ferritin concentrations), which help distinguish among anemias caused by iron deficiency, chronic disease, and thalassemia. 6
7 a. Ferritin is a very sensitive test for iron deficiency and in fact it goes down before iron levels or hemoglobin levels go down. b. Serum iron binding capacity is high in iron deficiency anemia as it reflects the amount of iron that can bind to transferrin (remember the empty chair analogy!) 2- Plasma unconjugated bilirubin, haptoglobin, and lactate dehydrogenase levels, which are abnormal in hemolytic anemias. 3- Serum and red cell folate and vitamin B12 concentrations, which are low in megaloblastic anemias. 4- Hemoglobin electrophoresis, which is used to detect abnormal hemoglobins. 5- Coombs test, which is used to detect antibodies or complement on red cells in suspected cases of immune hemolytic anemia Other tests will be described in the relevant sections, please see below for further description for the above- mentioned tests. CLASSIFICATION OF ANEMIA There are two classification systems for anemia: - The first one is based on etiology, anemia can be divided into o Anemia of decreased production in which anemia develops due to decrease synthesis of RBCs in the bone marrow o Anemia of increased peripheral removal of RBCs; this includes mechanisms in which RBCs are removed from the blood in a rate that exceeds the bone marrow capacity for compensation. o The best way to differentiate between the two is reticulocyte count (see below) - The second one is based on the size of the RBCs reflected by the MCV, o Microcytic: MCV is below reference range o Normocytic: MCV is within reference range o Macrocytic: MCV is above reference range. The first classification is a pathophysiologic one and is very important to understand the mechanism of anemia; it will be the basis of our lectures, the second classification is a clinical and will be vital when you assess your patients. 7
8 The role of reticulocyte count in differentiating anemia of decreased production from anemia of increased peripheral removal/destruction RBC production in the bone marrow starts as a stem cell that is capable of division and differentiation. The stem cell undergoes a series of differentiation steps in which it becomes more differentiated, synthesizes hemoglobin and loses the nucleus. The step that directly precedes the mature RBC is called a reticulocyte. Reticulocytes are larger than the mature RBC and appear a little bit darker as they contain remnants of RNA, the reticulocyte does not have a nucleus. Normally, the reticulocyte count should not exceed 1-2%. So how does the reticulocyte count help in classifying anemia? Well, if the cause of anemia is decreased production at the level of bone marrow, such as cases of leukemia, then ALL CELLS will decrease in number including reticulocytes, however if the anemia is caused by something removing RBCs from the peripheral blood, such as hemorrhage or hemolytic anemia, then the bone marrow will try to compensate for the RBC loss by increasing production, in this case, reticulocytes will not have enough time to mature in the bone marrow, and will be expelled to the periphery faster than normal, hence the number of reticulocytes will increase. In summary, low reticulocyte count means decrease production and high reticulocyte count means peripheral removal. As mentioned previously, the second classification looks at the MCV and divides anemia into three categories: - The most important causes of microcytic anemia are o Iron deficiency anemia o Thalassemia o Sideroblastic anemia o Lead poisoning - Macrocytic anemia is divided into two classes o Megaloblastic anemia: caused by B12 and/or folate deficiency o Nonmegaloblastic anemia: such as hypothyroidism, myeodysplastic anemia, and some cases of aplastic anemia 8
9 Please take a look at the following chart Please note the following - This chart combines both classifications - Most cases of micro- and macrocytic anemia are anemias of decreased production. - Normocytic anemia can be due to either peripheral removal or decreased production, the best way to differentiate is reticulocyte count. - Some disease can be normocytic or macrocytic, and some can be either normocytic or microcytic. 9
10 ANEMIA OF DECREASED PRODUCTION Many causes of anemia of decreased production exist; however, in this discussion only the most common entities will be covered. Specifically, iron deficiency anemia, anemia of chronic disease, megaloblastic anemia, anemia in liver disease, anemia of chronic renal disease, aplastic anemia and myelophthisic anemia. Before going with detail into these disease entities, a quick overview of iron metabolism is presented, it is very important to understand iron deficiency anemia and anemia of chronic disease. Approximately two thirds of iron is present in hemoglobin, another 6% is present in myoglobin. Around 25% of iron is present in storage and transport status, specifically, transferrin and ferritin. Transferrin is the major transporter of iron in the serum, and ferritin is the major storage protein for iron inside the cells, however, small amounts of ferritin are also present in the serum and are very helpful in the assessment of iron stores in the body (see below). Iron is present in the diet in two forms: heme iron and nonheme iron, heme iron is present in meat and poultry and is more readily absorbable than nonheme iron which is present in vegetables. Regulation of iron absorption occurs within the duodenum (see figure). After reduction by ferric reductase, ferrous iron (Fe 2+ ) is transported across the apical membrane by divalent metal transporter- 1 (DMT1). A second transporter, ferroportin, then moves iron from the cytoplasm to the plasma across the basolateral membrane. The newly absorbed iron is next oxidized by hephaestin and ceruloplasmin to ferric iron (Fe 3+ ), the form of iron that binds to transferrin. Both DMT1 and ferroportin are widely distributed in the body and are involved in iron transport in other tissues as well. As depicted in the figure (middle panel), part of the iron that enters enterocytes is delivered to transferrin by ferroportin, whereas the remainder is incorporated into cytoplasmic ferritin and is lost through the exfoliation of mucosal cells. The fraction of iron that is absorbed is regulated by hepcidin, a small peptide that is synthesized and secreted from the liver in an iron- dependent fashion. In general, high iron levels in the plasma enhance hepcidin production, whereas low iron levels suppress it. However, hepcidin production also is sensitive to inflammation and to factors released from erythroblasts in the bone marrow. Specifically, hepcidin levels rise in the face of systemic inflammation because of the direct effects of inflammatory mediators such as IL- 6 on hepatocytes, and in the setting of ineffective hematopoiesis, which is marked by increased numbers of erythroblasts in the bone marrow. Hepcidin circulates to the duodenum, where it binds ferroportin and induces its internalization and degradation. Thus, when hepcidin concentrations are elevated (see figure right panel), such as when serum iron levels are high or there is systemic inflammation, ferroportin levels fall and more iron is incorporated into cytoplasmic ferritin and is lost by excretion. Conversely, when hepcidin levels are low (see figure, left panel), such as when there is iron deficiency, ineffective hematopoiesis, or the genetic defects that lead to primary hemochromatosis, the basolateral transport of iron is increased. In iron 10
11 deficiency, the suppression of hepcidin is beneficial as it serves to help to correct the deficiency, but inappropriately low levels of hepcidin, as in primary hemochromatosis, eventually lead to systemic iron overload. The human body controls iron levels through absorption only as there is no mechanisms to control the excretion, iron is excreted in a fixed rate of approximately 1 to 2 mg/day through the shedding of mucosal and skin epithelial cells, and this loss must be balanced by the absorption of dietary iron, which is tightly regulated (see previous discussion). FIGURE: Regulation of iron absorption. Duodenal epithelial cell uptake of heme and nonheme iron is depicted. When the storage sites of the body are replete with iron and erythropoietic activity is normal, plasma hepcidin balances iron uptake and loss to maintain iron hemeostasis by downregulating ferroportin and limiting iron uptake (middle panel). Hepcidin rises in the setting of systemic inflammation or when iron levels are high, decreasing iron uptake and increasing iron loss by the shedding of duodenocytes (right panel), and it falls in the setting of low plasma iron or primary hemochromatosis, resulting in increased iron uptake (left panel) with reduced shedding. DMT1, Divalent metal transporter-1. 11
12 IRON DEFICIENCY ANENMIA Deficiency of iron is the most common nutritional deficiency in the world and results in clinical signs and symptoms that are mostly related to anemia. Four major mechanisms contribute to iron deficiency: 1- Chronic blood loss is the most important cause of iron deficiency anemia in the Western world. The most common sources of bleeding are the gastrointestinal tract (e.g., peptic ulcers, colon cancer, hemorrhoids) and the female genital tract (e.g., menorrhagia, metrorrhagia, endometrial cancer). 2- In the developing world, low intake and poor bioavailability because of predominantly vegetarian diets are the most common causes of iron deficiency. In the United States, low dietary intake is an infrequent culprit but is sometimes culpable in infants fed exclusively milk, in the impoverished, in the elderly, and in teenagers subsisting predominantly on junk food. 3- Increased demands not met by normal dietary intake occur worldwide during pregnancy and infancy. 4- Malabsorption can occur with celiac disease or after gastrectomy. Clinical findings: Iron deficiency anemia is insidious and asymptomatic and occasionally the apparent symptoms are those of the underlying disease (for example, excessive menorrhagia in a female). When symptoms develop, they are those of anemia (weakness, pallor etc). Additionally, spooning of the fingernails and pica (eating nonedible materials such as dirt, clay and paint) might develop. A rare syndrome characterized by iron deficiency, glossitis, esophageal webs and difficulty swallowing is known as Plummer- Vinson syndrome is well documented. (See figure) 12
13 Laboratory findings: In peripheral smears, red cells are microcytic and hypochromic (see figures). Diagnostic criteria include anemia, hypochromic and microcytic red cell indices, low serum ferritin and iron levels, low transferrin saturation, increased total iron- binding capacity, increased RDW and, ultimately, response to iron therapy. For unclear reasons, the platelet count often is elevated. Erythropoietin levels are elevated, but the marrow response is blunted by the iron deficiency; thus, marrow cellularity usually is only slightly increased. 13
14 The upper left image shows a completely iron depleted bone marrow The lower right image depicts a normal bone marrow with normal iron (blue) 14
15 Treatment is directed at replacing iron and most importantly at treating the underlying condition, treating the causative disease (colon cancer) is more vital than treating mild asymptomatic iron deficiency anemia. ANEMIA OF CHRONIC INLAMMATION Often referred to as the anemia of chronic disease, anemia associated with chronic inflammation is the most common form of anemia in hospitalized patients. It superficially resembles the anemia of iron deficiency but arises instead from the suppression of erythropoiesis by systemic inflammation. It occurs in a variety of disorders associated with sustained inflammation: 1- Chronic microbial infections, such as osteomyelitis, bacterial endocarditis, and lung abscess 2- Chronic immune disorders, such as rheumatoid arthritis and regional enteritis 3- Neoplasms, such as Hodgkin lymphoma and carcinomas of the lung and breast Pathogenesis: the key element in anemia of chronic disease is elevated hepcidin which results from increase serum levels of IL- 6. Hepcidin inhibits iron transportation from bone macrophages into erythroid precursor cells via downregulation of ferroprotein. Additionally, chronic inflammation suppresses erythropoietin production by the kidneys. The functional advantages of these adaptations in the face of systemic inflammation are unclear; they may serve to inhibit the growth of iron- dependent microorganisms or to augment certain aspects of host immunity. Clinical Features As in anemia of iron deficiency, the serum iron levels usually are low in the anemia of chronic disease, and the red cells may be slightly hypochromic and microcytic. Unlike iron deficiency anemia, however, storage iron in the bone marrow and serum ferritin are increased and the total iron- binding capacity is reduced. Administration of erythropoietin and iron can improve the anemia, but only effective treatment of the underlying condition is curative. 15
16 MEGALOBLASTIC ANEMIA Megaloblastic anemia is caused by deficiency of vitamin B12 and/or folate deficiency. B12 and folate are important cofactors in the synthesis of thymidine, one of the four building blocks of DNA. Rapidly dividing cells, especially the bone marrow cells, are more affected than other cells. The name megaloblastic refers to the morphologic changes seen in bone marrow; the erythroid precursor cells are larger than normal and show immature nuclei resulting from defective DNA synthesis, however, RNA and hemoglobin synthesis proceed normally, and hence, the cytoplasm shows normal maturation, this phenomenon is called nuclear- cytoplasmic asynchrony, meaning that the cytoplasm and nuclei are maturing in different rates. Additional morphologic findings include hypersegmented neutrophils (neutrophils with nuclei showing more than 4 segments) and abnormally shaped platelets. 16
17 Defective DNA synthesis results in increased apoptosis of the erythroid precursor cells and decreased cell division, which contributes to anemia. All bone marrow cells are affected, and it is common for those patients to present pancytopenia. In addition to the morphologic changes seen in the images above, the RBCs are usually oval shaped and markedly macrocytic (MCV more than 110). Folate deficiency Folate deficiency results from 1- Dietary: the most common cause, remember, the body storage of folate is sufficient for a couple of months only and if the diet is poor in folate, deficiency develops rapidly, in contrast to B12 (see below). 2- Increased demands during pregnancy and infancy. 3- Certain foods such as acidic foods and beans 4- Drugs such as phenytoin which interferes with absorption and methotrexate which interferes with metabolism. 5- Malabsorptive disorders, such as celiac disease and tropical sprue, that affect the upper third of the small intestine where folate is absorbed. Clinical manifestations Typically insidious and nonspecific, weakness, fatigue and shortness of breath if severe. Could be completely asymptomatic. Since the cells of the GI tract are rapidly dividing, symptoms related to the GI such as sore tongue can develop. Unlike vitamin B12 deficiency, no neurological symptoms are seen in the setting of folate deficiency. The diagnosis of a megaloblastic anemia is readily made from the examination of smears of peripheral blood and bone marrow. The anemia of folate deficiency is best distinguished from that of vitamin B12 deficiency by measuring serum and red cell folate and vitamin B12 levels. Vitamin B12 deficiency Dietary deficiency is an exceptionally rare cause of B 12 deficiency and is noted only among strictly vegan people who do not consume meat, milk, eggs or cheese. B12 stores in the body are sufficient for 5-20 years. Vitamin B12 deficiency results from diseases that interfere with its absorption: 17
18 1- Pernicious anemia: the most common cause, in pernicious anemia antibodies against the gastric parietal cells (the cells that produce intrinsic factor which is vital for B12 absorption) result in damage to these cells and decreased production of intrinsic factor 2- Gastrectomy(leading to loss of intrinsic factor producing cells) 3- Ileal resection(resulting in loss of intrinsic factor B12 complex absorbing cells). 4- Disorders that disrupt the function of the distal ileum (such as Crohn disease, tropical sprue, and Whipple disease). 5- In older persons, gastric atrophy and achlorhydria may interfere with the production of acid and pepsin, which are needed to release the vitamin B12 from its bound form in food Clinical manifestations In addition to the nonspecific symptoms seen in folate deficiency, patient with vitamin B12 deficiency might have neurological symptoms, including: 1- Peripheral neuropathy: numbness, parasthesia and feeling cold of the distal parts of the lower limbs. 2- Loss of position sense and ataxia 3- Neuropsychiatric manifestations including delusions, hallucinations, cognitive changes (like memory decline), depression, and dementia PLEASE REMEMBER - Folate administration in a patient with vitamin B12 deficiency can alleviate anemia but it worsens the neurological symptoms. - Sometimes in patients with vitamin b12 deficiency, neurological symptoms might develop without anemia. - Anemia responds dramatically to parenteral vitamin B12, however, the neurologic manifestations often fail to resolve Findings supporting the diagnosis of vitamin B12 deficiency are (1) low serum vitamin B12 levels, (2) normal or elevated serum folate levels, (3) moderate to severe macrocytic anemia, (4) leukopenia with hypersegmented granulocytes, and (5) a dramatic reticulocytic response (within 2 to 3 days) to parenteral administration of vitamin B12. Pernicious anemia is associated with all of these findings plus the presence of serum antibodies to intrinsic factor. 18
19 ANEMIA OF CHRONIC LIVER DISEASE Anemia in the setting of chronic liver disease (cirrhosis for example) results from several etiologis: Iron deficiency is the most common Hypersplenism Therapy related hemolytic anemia and suppression of EPO receptor Alcoholic- cirrhosis- induced folate deficiency Morphologically: anemia is characterized by the presence of spur cells (also known as acanthocytes) which are large erythrocytes covered with spikelike projections that vary in width, length, and distribution. ANEMIA OF CHRONIC RENAL DISEASE Anemia of chronic renal disease also stems from several etiologies: Decrease EPO production by the damaged kidney. High levels of inflammatory cytokines Hemolysis Chronic bleeding Folate deficiency in patients on dialysis. Morphologically anemia of chronic kidney disease are characterized by ecchinocytes (also known as Burr cells) which are RBCs with circumferential short, wide- based membrane projections. 19
20 APLASTIC ANEMIA Aplastic anemia refers to a syndrome of chronic primary hematopoietic failure and attendant pancytopenia (anemia, neutropenia, and thrombocytopenia). The bone marrow is completely devoid of hematopoietic cells and is filled with adipose tissue instead. 20
21 Etiology Most commonly is idiopathic. Known causes include drugs, chemical agents, infection, hereditary syndromes among others (see table) PATHOGENESIS: Two mechanisms: 1- An immune attack against the multipotent stem cells by T lymphocytes, this theory is supported by the fact the a sizable majority of patients respond well to anti- T cell immune therapy. 2- Stem cell intrinsic abnormality which results in decreased ability of stem cells to divide and differentiate and also increased early cell death. Clinical manifetations 1- Anemia due to decreased RBC production 2- Increased risk of infections due to neutropenia 3- Bleeding: petichiae and ecchymosis due to decreased platelets. Aplastic anemia does not cause splenomegaly; if splenomegaly is present, another diagnosis should be sought. Treatment is by anti- T cell immune therapy or by bone marrow transplantation. 21
22 MYELOPHTHISIC ANEMIA. Myelophthisic anemia is caused by extensive infiltration of the marrow by tumors or other lesions. It most commonly is associated with metastatic breast, lung, or prostate cancer. Other tumors, advanced tuberculosis, lipid storage disorders, and osteosclerosis may produce a similar clinical picture. The principal manifestations include anemia and thrombocytopenia; in general, the white cell series is less affected. Characteristically misshapen red cells, some resembling teardrops, are seen in the peripheral blood. Immature granulocytic and erythrocytic precursors also may be present (leukoerythroblastosis) along with mild leukocytosis. Treatment is directed at the underlying condition tear drop RBC 2-immature erythroid precursor cell 3-immture myeloid cell End 22
23 23
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