Iron Deficiency and Microcytic Anemias

Iron is an essential element for oxygen transport within hemoglobin. Oddly enough it is the element that is missed the most in regards to adequate intake and proper nutrition. Over 1.62 billion people in the world are effected by anemia, which is most commonly caused by iron deficiency. Iron deficiency can be caused by chronic blood loss, and is most common in women and teenagers from loss of blood due to menses. Iron loss leads to increased fatigue and depression, pallor, and dry and splitting hair. It can also lead to confusion cognitive effects. Hemoglobin is made of four polypeptide chains, two of which are alpha, and two are beta that come together to form a tetramer heme group with iron located in the middle. Ferrous iron within each heme molecule reversibly binds to one oxygen molecule. With iron deficiency, there becomes a hemoglobin deficiency. A decreased hemoglobin lowers oxygen-carrying capacity leading to anemia. Anemia by definition is a reduced oxygen-carrying ability. Tissue hypoxia can wreak havoc on almost every cell of the body, and can shift the oxygen dissociation curve in an unfavorable direction. The structure of hemoglobin and its function and key elements can be reviewed here.

To understand iron deficiency its important to recognize important aspects of iron metabolism and transportation in cells. Review the Iron Absorption and Metabolism article here for that information. There are also laboratory values that give a good picture of the iron status within the body that one should pay attention to. Transferrin; which is measured as the total iron binding capacity (TIBC) indicates how much or how little iron is being transported throughout the body. Serum iron is an important indicator of the tissue iron supply, and finally serum ferritin gives a picture of iron storage status within the bone marrow and cells.

Iron Deficiency Anemia

There are three stages within iron deficiency. Each comes with their own classic picture of laboratory results and worsen from stage to stage. In the first stage, there is storage iron depletion. This is mild and the patient may not even feel a difference physically. The patients hemoglobin is normal, normal serum iron, and TIBC. There is however decreased ferritin which indicates that there is decreased storage of iron. The second stage of iron deficiency is characterized by transport iron depletion. The hemoglobin may or may not be abnormal, but there is increased TIBC, and decreased serum iron. An increased TIBC, means that there are more substrate (iron) binding spots within the transferrin molecule. This implies that less iron is binding, which when coupled with a decreased serum iron makes sense. The patient may experience mild anemia which comes with increased fatigue and pallor. A peripheral blood smear will most often start to exhibit anisocytosis and poikilocytosis. These reference indicators represent abnormal sized red cells and abnormal shaped red blood cells respectively. A good indicator is an increased RDW, an increased RDW indicates some degree of anisocytosis. This is accurate because the red blood cell is realizing the loss of this oxygen-carrying capacity so its trying to release red blood cells as fast it can from the bone marrow to compensate for the loss, and as a result these red blood cells will appear smaller in diameter and hypochromic. Hypochromasia indicates that there is less hemoglobin within the cell and there is more of a central pallor. The thought is that even though there is less hemoglobin within each cell, if the bone marrow can produce more of these red blood cells than normal then that equals out. This leads to a microcytic anemia, micro meaning small. Stage three of iron deficiency is often referred to as functional iron deficiency. Within this stage there is an unmistakable decrease in hemoglobin, serum iron, and ferritin. There is also a large increase in TIBC.

The overall effect of iron deficiency anemia on the body and on the bone marrow is ineffective erythropoiesis. The red cell production within the bone marrow is compromised. As a result, the bone marrow becomes hypercellular with red cell precursors reducing the M:E (Myeloid:Erythroid) ratio.

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This picture depicts how a peripheral blood smear would illustrate iron deficiency anemia. The red cells are smaller and there is more of a central pallor to them, indicating a loss of hemoglobin. This is also called hypochromia.

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This picture depicts a normal peripheral blood smear. The red blood cells are larger in size and they have more color to them.

Anemia of Chronic Disease

Anemia of chronic disease is another form of microcytic anemia similar to iron deficiency anemia. It usually arises from a chronic infection or from chronic inflammation, but its also associated with some malignancies. A buildup in inflammatory cytokines alters iron metabolism. IL-6, which is an inflammatory cytokine inhibits erythrocyte production. It also increases hepcidin production. Hepcidin blocks iron release from the macrophages and the hepatocytes by down-regulating ferroportin. Without ferroportin there is no transportation of iron throughout the body and no production of hemoglobin or red blood cells. Laboratory findings will usually demonstrate low serum iron, low TIBC, low transferrin, and an increased to normal ferritin. The reticulocyte count is also normal, and sometimes increased. Reticulocytes are released from the bone marrow in times of red cell shortages to compensate.

This is just a brief overview of iron deficiency anemia and other microcytic anemias. This is just the beginning, follow and look forward to more in-depth reviews of each microcytic anemia. Key differences to look for is the TIBC value. In iron deficiency anemia the TIBC is increased and in anemia of chronic disease the TIBC is decreased. Ferritin is increased in anemia of chronic disease because the stored iron can’t be released from cells and the bone marrow due to the increased hepcidin production. Also the degree of anemia is mild compared to the more severe iron deficiency anemia.

 

 

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Iron Absorption and Metabolism

Iron is an essential element for almost all forms of life, but most important as an oxygen transporter. When iron is in its ferrous state, oxygen binds to it within the hemoglobin molecule allowing erythrocytes to circulate and deliver oxygen to all the human bodies cells and tissues. The human body also requires iron in order to obtain ATP from cellular respiration (Oxidative phosphorylation). Although iron is an essential element to the body, like anything in nature, too much of it can be toxic. Its ability to donate and accept electrons readily means that it can spontaneously catalyze the conversion of hydrogen peroxide into free radicals. Free radicals cause a wide array of damage to cellular structures and tissues. To minimize the chances of toxicity, almost every iron atom is bound to protein structures, an example being hemoglobin. The iron is bound to the globin protein. To learn more about the structure of hemoglobin, review the previous article written. There is tight regulation of iron metabolism that allows the body to remain in homeostasis. Understanding iron metabolism is important for understanding multiple diseases of iron overload, and iron deficiency.

Iron Absorption

Most of the bodies iron comes from dietary uptake. There is continuous iron recycling occurring within the body from the sequela of hemoglobin metabolism by the spleen. The macrophages of the reticuloendothelial system store iron from the process of breaking down engulfed red blood cells. Its stored as hemosiderin. Hemosiderin is just a defined deposit of protein and iron that occurs as a result of iron overload, either systemically or locally. The metabolic functions of iron depend on the ability to change its valence state from reduced ferrous state (Fe2+) to the oxidized ferric state (Fe3+). Ferrous iron in the lumen of the duodenum is transported across the luminal side of the enterocyte by a protein called divalent metal transporter-1 (DMT1). Once iron has been absorbed across the cell membrane of the enterocyte, it can either be stored by binding to apoferritin or the cell can release the iron through the help of another transporter called ferroportin. Ferroportin is the only know protein that exports iron across cell membranes. One of the ways that the human body manages iron homeostasis is by the production of hepcidin. When iron stores are adequate, the liver will produce hepcidin, which competitively binds to ferroportin and inactivates it. When iron stores begin to drop, suppression of synthesis of hepcidin allows ferroportin to transport iron again. Before iron is taken by ferroportin across the membrane, it must be converted to its ferric form. Hephaestin, another protein on the enterocyte cell membrane oxidizes iron as it exits to its ferric form (Fe3+). Once oxidized and in its ferric state, the iron binds to apotransferrin (ApoTf). This iron:apotransferrin complex is known as transferrin (Tf). Its important to note that two molecules of ferric iron can bind to one molecule of apotransferrin.

Mechanism-of-intestinal-iron-absorption-at-low-and-high-serum-iron-levels

Iron Uptake into Cells

Individual cells regulate the amount of iron they absorb to avoid adverse toxicity. Cells possess a receptor for transferrin (Tf), called transferrin receptor-1 (TfR1). The physiological pH of the plasma and extracellular fluid allow for a strong affinity to transferrin for TfR1. Through receptor mediated endocytosis transferrin saturates the TfR1 and once a critical mass has accumulated, endocytosis begins. The iron is passed into the cell into an endosome vesicle. Hydrogen ions are then pumped into the endosome and as a result the pH drops causing dissociation of the iron from the transferrin. Almost simultaneously the affinity for TfR1 to apotransferrin increases so it remains bound to the receptor while the iron remains free. The iron is then exported from the endosome vesicle into the cytoplasm by divalent metal transporter 1 (DMT1). The molecules of iron are then either stored, or transported into the mitochondria where they are incorporated into cytochromes or heme for the production of hemoglobin. While the iron is transported in the cytoplasm, the endosome fuses again with the cell membrane and in the extracellular space pH, TfR1 has a low affinity for apotransferrin so it dissociates and begins circulating again in the plasma for free transferrin. Again transferrin being a diiron:apotransferrin complex. Cells are able to store iron so they have a reserve if needed. Ferric iron (Fe3+) is stored in a protein called apoferritin. When iron binds to it it known as ferritin. Ferritin can be used at anytime during iron depletion by lysosomal degradation of the protein.

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Regulation

Just like hepcidin, there are other ways that the body maintains iron homeostasis. Transcription of TfR1 on the surface of the cells can either decrease or increase depending on iron stores within the cell. When iron stores are sufficient, production of TFR1 decreases, and vice versa. This is also useful in diagnosis of iron deficiency. Turns out there is a truncated form of TfR1 that circulates in serum as soluble transferrin receptors (sTfR). These sTfRs reflect the amount of tFR1 in the body. So in iron depletion there will be more circulating sTfRs indicating more production of TfR1 on the cells surface. A useful tool in the diagnosis of iron deficiency anemia.

Iron Recycling

When cells die, they are sequestered by the spleen and liver in which mechanisms salvage iron. These mechanisms are often referred to as the haptoglobin-hemopexin-methemalbumin system. Free hemoglobin in the plasma is quickly complexed with haptoglobin. By binding haptoglobin, the hemoglobin, and consequently, the iron avoid filtration by the glomerulus in the kidneys. This complex is taken up by macrophages, primarily those in the liver, spleen, bone marrow and even in the lungs. These macrophages express CD163, which is the haptoglobin scavenger receptor. The entire complex is internalized into the macrophage within a lysosome. Inside this lysosome, the iron is salvaged, the globin is catabolized as any protein would be, and the protoporphyrin is converted to unconjugated bilirubin. To learn more about the process of bilirubin metabolism, review the previous article. The haptoglobin is also degraded by the lysosome. The iron in free hemoglobin becomes oxidized to its ferric state (Fe3+), and as a result, forms methemoglobin. The heme (metheme) molecule of the free hemoglobin binds to hemopexin, preventing oxidative injury to the cells and tissues, as well as prevents loss of iron through glomerulus filtration. Albumin acts as a carrier for many proteins, including metheme. So albumin acts as a carrier for metheme to find hemopexin, which has a much higher affinity for the metheme itself. This allows for more rapid degradation of the toxic metheme.

There was a lot to learn in this article. Read carefully and go back and refer. I will try to highlight certain areas that I think are more important to the bigger picture. The next step is what happens in certain physiological disease states that leads to either iron overload or iron deficiency.

Polycythemia Vera

Polycythemia vera is an uncommon neoplasm or blood cancer where the bone marrow produces too many erythrocytes, megakaryocytes, and granulocytes, resulting in panmyelosis. The cancer is caused by a mutation in the JAK2 gene. Janus Kinase 2 (JAK2) is a non-receptor tyrosine kinase that plays a role in signaling in the type II cytokine receptor family. Members of that family include interferon receptors, GM-CSF receptor family, gp130 receptors, and the single chain receptors (EPO-R, etc). The function of those receptors are not important. The most important receptor for this article is the EPO-R receptor. The erythropoietin receptor (EPO-R) is a protein encoded by the EPOR gene that pre-exists in a dimerized state. When the ligand erythropoietin binds to the EPO-R receptor it induces a conformational change that results in the autophosphorylation of the JAK2 kinases. This establishes the function of EPO-R which is to promote proliferation and the rescue of erythroid progenitors from apoptosis. EPO-R induces JAK2-STAT5 signaling and with help from the transcription factor GATA-1 induces the transcription of the protein BCL-XL which is anti-apoptotic and promotes red cell survival.

In polycythemia vera (PV) there is a JAK2V617F mutation that causes independent continuous expression of the JAK2 kinase without erythropoietin (EPO) that acts on signaling pathways involving the EPO-R or hyperexpression in the presence of EPO. This causes increased gene expression for erythroid precursor cell proliferation and differentiation. It up regulates BCL-XL, which as mentioned above is an anti-apoptotic. This causes an abnormal accumulation of red cells in the peripheral blood. Its important to note that the accumulation of the red cells is due to lack of apoptosis, NOT because they are dividing quicker. Also there is a difference between primary PV and secondary PV. In primary PV there is a decreased expression of EPO, this is a compensation method for the body. As there is autophosphorylation of the EPO-Receptor, the body tries to reverse the process by down regulating the expression of erythropoietin (EPO). In secondary PV, there is normal to increased expression of EPO.

P-vera

Diagnosis

Diagnosis of PV according to the World Heath Organization (WHO) has to satisfy both major and minor criteria. The major criteria that has to be observed is a hemoglobin higher than 18.5 g/dL in men, and greater than 16.5 g/dL in women. There also has to be the presence of the JAK2 mutation. Minor criteria include presence of bone marrow hypercellularity demonstrating panmyelosis, serum EPO levels decreased, and a demonstration of endogenous erythroid colony growth in vitro. Meaning that there is presence of red cell growth in the laboratory using EPO from the patient, which assumes there is an issue with the downstream signaling of EPO, not EPO itself.

Laboratory results illustrate an increased hemoglobin, hematocrit, and MCV. There is an increased red cell count, platelet count, and white blood cell count. The leukocyte alkaline phosphatase is also increased. Its important to know that although the platelet count is increased, there is also an altered function of the platelets. The erythrocyte sedimentation rate will be decreased due to the decrease in the zeta potential. The zeta potential is the electrokinetic potential between the red cells that stops them from stacking or from sticking to one another. One classic characteristic of PV is erythromelalgia. This is a burning sensation in the pain and feet, with a reddish or bluish discoloration. This is caused by an increased platelet agglutination, from being dysfunctional that results in microvascular blood clots.

Treatment

If untreated, PV can be fatal. Although the disease can’t be cured, it can be controlled and the life expectancy has risen with modern advances in medicine. Phlebotomy is recommended to reduce the hemoglobin and hematocrit levels, but can induce iron deficiency anemia if not monitored. Low dose aspirin is prescribed to reduce the risk of thrombotic events. The accumulation of the red cells increases the risk for the patient to develop thrombotic events because the blood is “thick”. Chemotherapy can be used, but is not normally indicated, unless therapeutic phlebotomy is unable to maintain a normal hemoglobin or hematocrit or when there is significant thrombocytosis. It is dangerous because of the risk for transformation to acute myeloid leukemia (AML).

To recap; its important to know the mutation in the JAK2 kinase that induces polycythemia vera. Although this mutation is demonstrated in 90% of cases, its possible that its absent. Panmyelosis and elevation of RBC indices is a diagnostic finding. Its important to know the major and minor criteria for the diagnosis of PV. Treatment is therapeutic phlebotomy and chemotherapy in rare cases, only when prior treatment has failed.

Thalassemias

PIT11

The major hemoglobin that is present in adults is hemoglobin A (HbA). This is a heterotetramer that consists of one pair of alpha-globin chains and one pair of beta-globin chains. Alpha-globin chains are encoded by two copies of the alpha gene present on chromosome 16. Beta-globin chains are driven by one gene on chromosome 11. Normally there is tight regulation of the production of alpha and beta-globin chains and the ratio of production, but sometimes that regulation can be interrupted. This offsets the balance of globin chains being produced. These types of hematological disorders are coined thalassemias. They are a quantitative defect characterized by reduced or absent production of one and rarely two of the globin chains.

Alpha thalassemia is largely due to the inadequate production of alpha globin chains, which leads to an excessive production of either gamma-globin chains or beta-globin chains. In the fetus alpha thalassemia leads to excess gamma chains and in adults it largely leads to excess beta chains. In neonates the absence of alpha-globin chains is incompatible with life, leading to hydrops fetalis or hemoglobin Barts and absolute death after delivery. Hb Barts cannot deliver the oxygen to the tissues because its affinity to oxygen is too high. The hydronic state is reflected in the fetus by heart failure and massive total body edema. Excess beta-globin chains are capable of forming homotetramers and precipitate that leads to a variety of clinical manifestations.

Beta thalassemia is an inherited hemoglobinopathy in which production of beta-globin chains is impaired. There are different classifications corresponding to the degree of reduction in the beta chains. Beta thalassemia major is due to mutations that completely stop all production of beta-globin chains. These are individuals who are homozygous for the disease. They lose the ability to make HbA and because of this will experience severe manifestations and are transfusion-dependent for the rest of life. Symptoms typically begin during late infancy (6-12 months), but some newborns are asymptomatic because the major hemoglobin in newborns is HbF (4A:4G) which is constructed by gamma-globin chains and not beta-globin chains. Beta-thalassemia major presents with pallor, jaundice, and bilirubin in the urine which indicates hemolysis. Hepatosplenomegaly is present as well as heart failure. Failure to thrive and recurrent infections are also other signs. There is so much hemolysis because of the faulty hemoglobin present in the red cells that the bone marrow can’t keep up with production so extra medullary hematopoiesis occurs that results in skeletal abnormalities in the face and long bones. Iron overload is often a symptom of late untreated disease which can affect almost every organ in the body. Mortality is upwards of 85% by age five if untreated. If treated the survival rate is only 60 years of age if lucky.

Beta thalassemia major is also called transfusion-dependent beta thalassemia. There is also a subtype called non-transfusion-dependent beta thalassemia otherwise known as beta thalassemia intermedia. These individuals present with a less severe phenotype of the disease. There is significant variability with the clinical findings in individuals with beta thalassemia intermedia; from osteoporosis to thrombosis to diabetes mellitus. Some individuals will develop hepatosplenomegaly and extramedullary hematopoiesis and some won’t. Also some individuals will have to become transfusion-dependent, but that is typically in the late decades of life.

Anemia is a severe clinical manifestation of both alpha and beta thalassemia. The pathophysiology of beta thalassemia causes excess alpha-globin chains to precipitate in the developing erythrocytes in the bone marrow. This causes inclusion bodies. The inclusion bodies create oxidative stress and damages the cellular membranes. Apoptosis gets activated downstream and the red cell precursors are subsequently phagocytized and destroyed in the bone marrow by activated macrophages. This is also called ineffective erythropoiesis. The bone marrow in an effort to compensate releases these red cell precursors into the peripheral blood riddled with these inclusion bodies. These cells are subsequently sequestered by extravascular hemolysis by the RES which  further contributes to the anemia. The red cells that survive are microcytic and hypochromic and have a significantly shortened life span. Severe tissue hypoxia is seen due to the increased HbF as a compensatory mechanism. HbF has an increased affinity for oxygen and causes a shift to the left on the oxygen dissociation curve.

Typical laboratory findings for an individual with beta thalassemia is a slightly decreased red cell count and a marked decrease in hemoglobin of usually about 2-3 g/dL (12.5-16.5 g/dL). There will be marked anisocytosis (microcytosis) and poikilocytosis, target cells, basophilic stippling, slight increase in reticulocytes and nucleated red cells.

The pathophysiology for anemia associated with alpha thalassemia is associated with precipitation of HbH. Remember HbH is formed when there is decreased production of the alpha-globin chains so there is an excess of beta-globin chains. The precipitation of HbH creates inclusion bodies, typically called Heinz Bodies. These inclusions are recognized by the RES and remove the red cells via extravascular hemolysis.

Laboratory findings for an individual with alpha thalassemia is very similar to that of an individual with beta thalassemia. Decreased hemoglobin, marked anisocytosis (microcytosis) and poikilocytosis, target cells, basophilic stippling, and reticulocytes and NRBCs. The HbH inclusions can be see seen using a cresyl blue stain.

Thalassemias are a quantitative hemoglobinopathy meaning that there is a deficiency or an excess of production of globin chains leading to clinical manifestations. They are inherited and some subtypes can significantly elevate mortality. It is important to diagnose early and to treat early.

 

 

 

Non-Malignant Leukocyte Disorders

Non-Malignant simply means that it is localized to the leukocytes. Leukocytes are another name for the white blood cells, more specifically in the case of these disorders, the granulocytes. These disorders are fairly uncommon and are inherited. The following are ones that are found to distinct morphological features and affect the granulocyte functionality.

Alder Reilly Anomaly

Alder Reilly Anomaly is a recessive trait defect that causes incomplete degranulation of mucopolysaccharides. Large, darkly staining metachromatic cytoplasmic granules which can be seen and are partially digested mucopolysaccharides. These granules are characteristically referred to as Alder Reilly bodies. These can sometimes resemble toxic granulation, but it is important to note that in toxic granulation neutropenia, dohle bodies, and a left shift is seen. In Alder Reilly Anomaly none of those are present. Its also important to mention that the functionality of the granulocytes is not impaired.

Alder Reilly

Pelger Huet Anomaly

Pelger Huet Anomaly is an autosomal dominant syndrome characterized by decreased nuclear segmentation. This is caused by a mutation in the Lamin B receptor gene. Lamin B is an inner nuclear membrane protein that plays a role in normal leukocyte nuclear shape change during maturation. Morphological changes include hyposegmented neutrophils or neutrophil lobes connected by a thin nuclear filament. Pseudo or acquired PHA can be observed in the granulocytes in individuals with MDS, AML, or chronic myeloproliferative neoplasms.

Pelger Huet

Chediak Higashi Syndrome

Chediak Higashi Syndrome is characterized by an abnormal fusion of granules. These present as large and are dysfunctional. This is caused by a mutation in the LYST, or CHS1 gene that encodes for proteins involved in vesicle fusion or fission. The mutated protein causes loss of lysosomal movement and loss of phagocytosis. Thus leaving the individual susceptible to an increased number of infections without the innate immune system to fight them off. One of the characteristic findings is neutropenia.

CHA

May-Hegglin Anomaly

May-Hegglin Anomaly is a rare autosomal dominant platelet disorder that is characterized by variable thrombocytopenia, giant platelets, and dohle bodie like inclusions in the granulocytes. MHA is caused by a mutation in the MYH9 gene that causes a dysfunctional and disarray production of myosin heavy chains type IIa which affects the megakaryocytic maturation process as well as platelet fragmentation. Though most cases are clinically asymptomatic, the individual may present with mild bleeding tendencies.

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Chronic Granulomatous Disease

In CGD, mutations in proteins that make up the NADPH oxidase complex. The mutations lead to failure of the phagocytes to generate the oxygen-dependent respiratory burst following phagocytosis. Normal phagocytosis of a microorganism leads to phosphorylation of cytosolic P47 and P67. Antibacterial neutrophil elastase and cathepsin G from the primary granules and cytochrome complex gp91 and gp22 from the secondary granules migrate to the phagolysosome. NADPH oxidase is formed when P47 and P67 combine with P40, RAC2, and the cytochrome complex. Majority of cases of CGD is due to mutations in P47 or gp91.

Leukocyte Adhesion Disorders

Normal recruitment of leukocytes to a site of inflammation involves capture of leukocytes from peripheral blood, followed by a process known as rolling along a vessel wall. Rolling involves binding of integrins to endothelial cell receptors which is high-affinity which ultimately leads to diapedesis of leukocytes into tissues from peripheral blood. With Leukocyte Adhesion disorders there are mutations that result in the inability of neutrophils and monocytes to adhere to endothelial cells, and the consequence is potentially fatal bacterial infections.

Leukocyte Adhesion Disorder I is caused by a mutation in the genes responsible for B2 integrin subunits. This leads to a decreased amount of the truncated form of the B2 integrin which is essential for endothelial cell adhesion. Patients typically present with neutrophilia, lymphadenopathy, splenomegaly, and characteristic skin lesions.

Leukocyte Adhesion Disorder II is caused by a mutation in the SLC35C1 gene. This gene encodes for a fucose transporter that moves fucose from the endoplasmic reticulum to the Golgi region. Fucose is needed for the synthesis of selectin ligands. The defective fucose transporter leads to the inability to produce functional selectins and causes defective leukocyte recruitment and reoccurring infections. LADII is much more rare than LADI. Clinical presentation is growth retardation, coarse facial features, and other physical deformities.

Leukocyte Adhesion Disorder III is even more rare than LADII and is caused by a mutation in the Kindlin-3 gene. The mutations impair leukocyte rolling and activation of B integrin. With LADIII there is also decreased platelet integrin GPIIbIIIa resulting in bleeding similar to that of Glanzmann Thrombasthenia.

-Caleb