ANEMIA AND HEMOGLOBINOPATHIES

• ACRONYMS

• AHA - Autoimmune hemolytic anemia
• AI - Anemia of inflammation
• C3 - Complement component 3
• CAHA - Cold autoimmune hemolytic anemia
• CDC - Centers for Disease Control and Prevention
• CHr - Content of hemoglobin in reticulocytes
• CKD - Chronic kidney disease
• DIC - Disseminated intravascular coagulation
• ESA - Erythropoietin-stimulating agents
• Hg - Hemoglobin
• HUS - Hemolytic uremic syndrome
• IRIDA - Iron-refractory iron deficiency anemia
• ITP - Immune thrombocytopenia
• MCV - Mean corpuscular volume
• PNH - Paroxysmal nocturnal hemoglobinuria
• RBC - Red blood cell
• SLE - Systemic lupus erythematosus
• TCD - Transcranial doppler ultrasound
• TSAT - Transferrin saturation
• TTP - Thrombotic thrombocytopenic purpura
• USPSTF - U.S. Preventive Services Task Force
• VTE - Venous thromboembolism
• WAHA - Warm autoimmune hemolytic anemia

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• DEFINITIONS

• Acanthocytes (spur cells) - acanthocytes are RBCs that have spikes along their membrane at irregular intervals. Acanthocytes occur because of abnormalities in the composition of the cell membrane. Acanthocytes may be seen in a number of conditions including liver disease (increase in membrane lipids) and vitamin deficiencies. (acanthocyte illustration)

• Anisocytosis - anisocytosis means the observed RBCs have varying sizes. Anisocytosis is common in anemia. The red cell distribution width (RDW) is a measure of anisocytosis with higher values indicating greater anisocytosis.

• Antibody screen - an antibody screen is a test that detects antibodies to surface antigens on RBCs. Antibodies to the main A and B red blood cell antigens occur naturally depending on a person's blood type. Antibodies to other RBC antigens occur through exposure to foreign RBCs, primarily through blood transfusions or pregnancy (mother-baby incompatibility).

• Apoferritin - apoferritin is a protein that resides in cells. It's primary function is to bind iron that has entered the cell at which point it becomes ferritin. Ferritin is the main storage form of iron accounting for 15 - 30% of total body iron.

• Apotransferrin - apotransferrin is a protein that binds iron in the plasma and intestines. The apotransferrin-iron complex is called transferrin, and it is the main form in which iron is transported around the body. Apotransferrin is mixed with bile and secreted into the small intestine where it binds free iron and iron-containing compounds like hemoglobin and myoglobin in meat. The apotransferrin-iron complex (transferrin) is then absorbed by the intestine. See iron homeostasis below.

• Basophilic stippling - "basophilic" means something that is readily stained with basic dyes. In histological terms, "stippling" means small dots. Basophilic stippling is a term that means small dots of basophilic ribosomes and RNA are speckled throughout an erythrocyte. Basophilic stippling occurs when there is incomplete ribosome degradation during the latter stages of erythropoiesis, which may occur in heavy metal toxicity (e.g. lead poisoning) and hemoglobinopathies such as thalassemia and sickle cell disease. (basophilic stippling illustration) [30]

• Bilirubin - bilirubin is a compound formed from the metabolism of hemoglobin. After macrophages consume hemoglobin, they break it down into iron and bilirubin. Bilirubin is released back into the bloodstream where it is removed by the liver and secreted into the bile. See RBC life cycle below.

• Bite cells (also called blister cells) - bite cells are RBCs with irregular membranes. Bite cells are formed when macrophages remove Heinz bodies from RBCs. Bite cells are a sign of G6PD deficiency. (Heinz body and bite cell illustration)

• Blood - blood is composed of plasma and formed elements (e.g. WBCs, RBCs, platelets). Plasma is blood minus all the formed elements, and serum is plasma minus the clotting factors. The average adult has 5 liters of blood.

• Carbon monoxide - carbon monoxide is a gas that binds almost irreversibly to hemoglobin and renders it unable to carry oxygen. Carbon monoxide poisoning is treated acutely with high-flow oxygen.

• Cold agglutinins - cold agglutinins are IgM antibodies that bind the I/i antigen on RBCs. They are most reactive between 39 and 70° F, hence the name "cold." If their concentration is high enough, they can cause RBC agglutination and fixation of C3 to the RBC membrane. C3-tagged RBCs may undergo intravascular hemolysis if the complement system is completed, but more commonly, they are removed by macrophages in the spleen and liver. Cold agglutinin antibodies are present at low titers in almost all humans, and they only become clinically significant at higher titers. Cold agglutinins may form secondary to an underlying condition (e.g. cancer, Mycoplasma pneumonia, mononucleosis), or they may be secondary to a clonal low-grade B-cell lymphoproliferative disorder. See cold autoimmune hemolytic anemia for more. [58]

• Direct Coombs test (direct antiglobulin test) - the direct Coombs test is an assay that looks for antibodies (IgG, "gamma globulins") and complement components (C3, "beta globulins") attached to RBCs. IgG attached to RBCs is consistent with WAHA. C3 attached to RBCs can occur with WAHA, but it is more common in CAHA. CAHA is caused by IgM antibodies that attach to the surface of RBCs and fix complement (C3). IgM can detach from RBCs and is not commonly detected on the direct antiglobulin test, but the presence of C3 fixation is a strong indicator of the presence of IgM antibodies. These autoimmune antibodies can lead to hemolysis and RBC destruction. See autoimmune hemolytic anemia for more.

• Deoxyhemoglobin - deoxyhemoglobin is hemoglobin that does not have oxygen bound to it.

• Echinocytes (Burr cells) - echinocytes are RBCs that have regularly spaced spikes along their membrane. Echinocytes occur when plasma abnormalities affect membrane morphology. Echinocytes can be seen in kidney disease, liver disease, and as an artifact of treating blood samples with EDTA to prevent clotting. (echinocyte illustration)

• Erythropoietin - erythropoietin is a hormone that stimulates RBC production in the bone marrow. 90% of erythropoietin is produced in the kidneys and 10% in the liver. Erythropoietin release is stimulated by tissue hypoxia, and it takes about 5 days after an increase in production before new RBCs are seen.

• Ferritin - ferritin is formed when iron binds the protein apoferritin inside of cells. Ferritin is the main storage form of iron iron accounting for 15 - 30% of total body iron. The serum ferritin level is directly proportional to tissue concentrations which makes it a good measure of total body iron stores. Ferritin is also an acute phase reactant and may be elevated in inflammatory conditions. See ferritin for more.

• Folate/Folic acid (Vitamin B9) - folate is a naturally-occurring vitamin found in dark leafy green vegetables, citrus fruits, and liver. Folic acid is a synthetic version of folate that is found in supplements and fortified foods such as breads and cereals. Folic acid is essential for the production of DNA. Folic acid deficiency causes RBCs to develop abnormally leading to enlarged RBCs called macrocytes. Macrocytes carry oxygen normally, but they are not as pliable as normal RBCs and their lifespan is decreased by 30 - 50%. See folic acid deficiency for more.

• Haptoglobin - haptoglobin is a protein produced by the liver that binds directly to hemoglobin in the blood and prevents the release of highly reactive heme. Haptoglobin-hemoglobin complexes are quickly removed by macrophages. Elevated LDH levels in combination with a haptoglobin < 25 mg/dL is 95% specific for the presence of hemolysis while a normal LDH and haptoglobin > 25 mg/dL are 92% sensitive for the absence of hemolysis. Low haptoglobin levels may also be seen in vigorous exercise, liver disease, transfusion therapy, and ahaptoglobinemia, a rare genetic disorder that affects 0.1% of whites and as many as 4% of blacks. Haptoglobin is also an acute phase reactant, and it may be normal or elevated despite significant hemolysis in patients with infections and other inflammatory states.

• Heinz bodies - Heinz bodies are clumps of denatured hemoglobin that appear in RBCs. They are not seen on standard blood smears, but can be visualized with supravital staining. Heinz bodies form under conditions that cause oxidative stress (e.g. G6PD deficiency), and they often lead to hemolytic anemia. When macrophages remove Heinz bodies from RBCs, the RBCs can develop an irregular border which gives them the name "bite cells" or "blister cells." (Heinz body and bite cell illustration)

• Hemosiderin - hemosiderin is an insoluble storage form of iron that normally makes up a small percentage of iron stores. If apoferritin stores are exhausted, hemosiderin production may increase and large clusters may form in cells. If these clusters are in the skin, something called "hemosiderin staining" can occur in which the skin appears hyperpigmented or darkened. Hemosiderin staining of the skin may occur in areas of trauma, bleeding, edema, and ulceration.

• Hemosiderinuria - in severe hemolysis, urinary hemoglobin is reabsorbed by renal tubular cells and processed to hemosiderin. Hemosiderinuria is the presence of hemosiderin in sloughed renal epithelial cells.

• Hepcidin - hepcidin is a hormone released by the liver that is involved in iron homeostasis. Hepcidin blocks iron absorption in the intestines and inhibits iron release from the liver and macrophages. Hepcidin levels increase in response to high levels of circulating iron and elevated ferritin stores, and release is suppressed by tissue hypoxia, erythropoiesis, low levels of circulating iron, and decreased ferritin. Hepcidin is also an acute phase reactant, and conditions causing prolonged inflammation can raise levels and lead to anemia of chronic inflammation.

• Howell-Jolly bodies - Howell-Jolly bodies are nuclear remnants (clusters of DNA) seen in circulating RBCs. Under normal circumstances, RBCs expel all of their nucleus before they leave the bone marrow, but a small portion may remain. The spleen removes any remaining nuclear remnants from circulating RBCs. Howell-Jolly bodies are seen in patients with hyposplenia and asplenia. They may also be present in megaloblastic anemia and hemolysis. (Howell-Jolly body illustration)

• Inclusion bodies - inclusion bodies are clumps of precipitated proteins that form inside RBCs. They may occur from abnormal hemoglobin, lead poisoning, and abnormal erythropoiesis.

• Iron - iron is an element found throughout the body that has many important functions. Iron binds loosely to oxygen in hemoglobin so that RBCs can transport oxygen throughout the body. Iron is also found in myoglobin (protein that stores oxygen in muscle cells), and it plays an important role in enzymatic reactions (e.g. cytochromes, catalase, peroxidase). Iron is stored in tissues throughout the body, and excess iron is primarily stored in the liver. See iron homeostasis below.

• Lactate dehydrogenase (LDH) - lactate dehydrogenase is an enzyme involved in anaerobic metabolism that is found in almost every cell in the body. When cells are damaged or destroyed, they release LDH. An elevated LDH level is a sensitive measure of RBC hemolysis, but it is not specific. LDH enzymes have subtypes, and subtypes 1 and 2 are seen in RBCs. Subtypes 1 and 2 are also seen in the kidneys, stomach, pancreas, and heart muscle. LDH levels can be used in combination with haptoglobin to detect hemolysis. Elevated LDH with a haptoglobin < 25 mg/dL is 95% specific for the presence of hemolysis while a normal LDH and haptoglobin > 25 mg/dL are 92% sensitive for the absence of hemolysis.

• Mean corpuscular volume (MCV) - the MCV is the average volume of RBCs in a blood sample. It is typically calculated by a machine called a hemolyzer. A normal MCV is 80 - 95 fL in adults.

• Methemoglobinemia - methemoglobin is hemoglobin with the iron in the ferric state (Fe⁺³) as opposed to the normal ferrous state (Fe⁺²). Methemoglobin cannot bind oxygen, and it causes the remaining subunits of normal hemoglobin to bind oxygen more tightly; this prevents the delivery of oxygen to tissues. Under normal conditions, methemoglobin makes up < 1% of hemoglobin. Levels above 1% are abnormal and consistent with methemoglobinemia. Methemoglobinemia can be congenital or it can be acquired from certain chemicals and/or medications (e.g. nitrates, local anesthetics, antibiotics, antimalarials). Methemoglobinemia is typically treated with methylene blue, a dye which rapidly converts methemoglobin to hemoglobin.

• Oxyhemoglobin (HbO₂) - oxyhemoglobin is hemoglobin that has oxygen bound to it

• Plasma - plasma is the liquid component of blood that does not contain the formed elements (e.g. RBCs, WBCs, platelets). The average adult has about 2.75 liters of plasma. Plasma is 93% water and the rest is dissolved or suspended solutes. Plasma must be frozen to preserve the clotting factors.

• Plasmapheresis - plasmapheresis is a procedure where a patient's plasma is exchanged for donor plasma

• Poikilocytosis - poikilocytosis is a term that means irregularly-shaped RBCs. Poikilocytosis can occur in a number of disorders including anemia and thalassemia to name a few.

• Polychromasia - polychromasia is a term that means there is a abnormally high number of reticulocytes in the circulation

• Red blood cells (RBCs) - RBCs (also called erythrocytes) are a type of blood cell that have the primary function of transporting hemoglobin to body tissues so that oxygen may be delivered to the cells. RBCs also contain carbonic anhydrase, an enzyme that serves as the main acid-base buffer in the body. Healthy red blood cells are very pliable and are able to squeeze their way through capillaries. All bones produce RBCs in their bone marrow up until the age of 5 years, after which, production primarily occurs in the ribs, sternum, pelvis, and iliac crest. Unlike other cells, RBCs do not have a nucleus or other organelles. See RBC life cycle below.

• Reticulocyte - reticulocytes are immature RBCs that form in the bone marrow and are released into the circulation. Reticulocytes circulate for 1 day before they become mature RBCs. Histologically, reticulocytes are distinguishable from mature RBCs by the persistence of a small amount of basophilic material consisting of cytoplasmic organelles. Under normal conditions, the percent of RBCs that are reticulocytes in adults is 0.5 - 2.5%. The absolute reticulocyte count is found by taking the percentage of reticulocytes and multiplying by the RBC count. (reticulocyte illustration)

• Reticulocyte hemoglobin content (CHr) - the reticulocyte hemoglobin content (also called content of hemoglobin in reticulocytes (CHr)) is the amount of hemoglobin observed in circulating reticulocytes. It is reported in picograms and normal ranges vary by age. In adults, levels > 28 pg typically reflect adequate iron available for erythropoiesis. Since reticulocytes represent the newest RBCs in the circulation with a lifespan of 1 - 2 days, CHr is considered a more acute measure of hematopoietic-available iron than ferritin and TSAT. CHr can also be used to predict responses to IV iron therapy and has been shown to be more accurate than ferritin and TSAT in some studies. [34]

• Reticulocyte index - the reticulocyte index is the ratio of the observed absolute reticulocyte count to the normal reticulocyte count. Reticulocyte index = [observed reticulocyte count] / [normal absolute reticulocyte count] (online calculator). A reticulocyte index ≥ 2 is considered an appropriate marrow response to anemia. An index < 2 is considered inappropriate and suggests conditions suppressing erythropoiesis.

• Reticulocyte production index (RPI) - the RPI is a measure of reticulocyte production that adjusts for the level of anemia. When erythropoietin stimulates RBC production, it causes the early release of reticulocytes from the bone marrow into the circulation. Under normal conditions, reticulocytes in the circulation mature into erythrocytes in 1 day. When erythropoietin-stimulated early release occurs, reticulocyte maturation can last up to 2.5 days. Prolonged maturation increases the number of circulating reticulocytes, but this does not reflect a true increase in erythropoiesis. The RPI corrects for the shift in maturation and provides a more accurate measure of marrow response. An RPI > 3 is considered an appropriate marrow response to anemia and an RPI < 2 is considered inappropriate. The RPI can be calculated with one of the two following formulas: RPI = [Reticulocyte %] X [Hg (observed) / Hg(normal)] X 0.5 or RPI = [reticulocyte index] / [expected maturation rate in days] using the following:
• Hg 10 - 13 g/dl: 1.5 days
• Hg 7 - 10 g/dl: 2 days
• Hg 3 - 7 g/dl: 2.5 days

• Schistocytes - schistocytes are fragments of RBCs formed when the cells are damaged or destroyed. Conditions where schistocytes are seen include DIC and TTP (microvascular fibrin strands shear RBCs), hemolytic anemia, and mechanical heart valves.

• Serum - serum is plasma minus the clotting factors. Serum contains electrolytes, proteins, immunoglobulins, hormones, and anything else not involved in coagulation.

• Sideroblasts - sideroblasts are erythroid precursor cells that have a ring of iron-laden mitochondria around at least one-third of the cellular nucleus. Iron-laden mitochondria form secondary to abnormalities in heme synthesis. See sideroblastic anemia. (sideroblast illustration) [32]

• Spherocytes - spherocytes are RBCs with defective membranes that allow sodium and water to enter the cell. This causes the RBC to assume the shape of a sphere as opposed to the typical round, biconcave shape. Spherocytes are caused by hereditary disorders of RBC membrane formation (hereditary spherocytosis), and they are also seen in hemolytic anemia when complement fixation creates holes in the membrane. (spherocyte illustration)

• Target cells - target cells are RBCs that have a central concentration of hemoglobin surrounded by a pale ring. Target cells form when there is a disproportionate increase in the ratio of cell membrane area to cell contents. The increased ratio can occur from a decrease in hemoglobin (e.g. iron deficiency anemia) or from an increase in cell membrane surface area (e.g. liver disease, splenectomy). In liver disease, cholesterol and phospholipids deposit in the surface of RBCs causing their surface area to increase. The spleen typically removes lipids from the maturing RBC membrane, so its absence (e.g. splenectomy) can lead to increased surface area as well.

• Transferrin - transferrin is formed when iron binds to the protein apotransferrin. Transferrin transports iron through the blood to various tissues in the body. Iron is loosely bound to transferrin so it is easily released to cells that need iron. Excess iron is primarily stored in liver cells. See iron homeostasis below.

• Urobilinogen - urobilinogen is formed in the intestine from the bacterial metabolism of bilirubin. Some urobilinogen is excreted in the feces and some is reabsorbed and excreted by the kidneys. Elevated urinary urobilinogen can occur under conditions of increased hemoglobin metabolism (e.g. hemolytic anemia, clot reabsorption) and liver inflammation (e.g. cirrhosis, hepatitis). Low levels of urinary urobilinogen may occur with hepatic or biliary obstruction. See iron homeostasis below.

• Vitamin B12 - vitamin B12 is essential for the production of DNA. Vitamin B12 deficiency causes RBCs to develop abnormally leading to enlarged RBCs called macrocytes. Macrocytes carry oxygen normally, but they are not as pliable as normal RBCs and their lifespan is decreased by 30 - 50%. The stomach produces a glycoprotein called intrinsic factor that binds and protects Vitamin B12 and makes it available for absorption in the ileum. Stomach conditions that affect intrinsic factor production (e.g. gastritis, gastric surgery) can lead to decreased Vitamin B12 absorption. Only 1 - 3 micrograms/day of Vitamin B12 are required to maintain normal RBC production, so it can take 3 - 4 years of deficient absorption to affect RBC production. Vitamin B12 is found in animal products (meat, eggs, dairy) so vegetarians are at risk of deficiency. See vitamin B12 deficiency for more.

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• EVALUATING ANEMIA

• Overview
• The cause of anemia is obvious in many cases but can be challenging to identify in others, especially when it is multifactorial. There are no professional guidelines that provide a straightforward diagnostic approach to anemia. The steps below are derived from review articles and offer a starting point that can be tailored to the individual. It's important to keep in mind that even after an extensive workup, the etiology of anemia will remain unknown in a significant number of cases.

• References [25,52]

Evaluating anemia
WHO definition of anemia Males: Hemoglobin < 13 g/dl Females: Hemoglobin < 12 g/dl
MCV is low (< 80 fL) Check iron studies Ferritin < 30 mcg/L and/or TSAT < 16% - iron deficiency is present. See iron deficiency anemia below. Ferritin 30 - 100 mcg/L and/or TSAT > 16% Consider iron deficiency anemia and/or anemia of inflammation. A high TIBC is consistent with iron deficiency, and a low TIBC is consistent with anemia of inflammation. If both conditions are present, the TIBC is not helpful. Consider alpha thalassemia and beta thalassemia in susceptible populations. A trial of oral iron therapy may be attempted. If a significant response occurs after 4 weeks, iron deficiency is the likely predominant cause. Ferritin > 100 mcg/L and/or TSAT > 20% Consider alpha thalassemia and beta thalassemia in susceptible populations.
MCV is normal (80 - 100 fL) Check iron studies and reticulocyte count Ferritin < 30 mcg/L and/or TSAT < 16% - iron deficiency is present. See iron deficiency anemia below. Ferritin > 30 mcg/L and/or TSAT > 16% Consider iron deficiency anemia and/or anemia of inflammation. A high TIBC is consistent with iron deficiency, and a low TIBC is consistent with anemia of inflammation. If both conditions are present, the TIBC is not helpful. A trial of oral iron therapy may be attempted. If a significant response occurs after 4 weeks, iron deficiency is the likely predominant cause. If GFR is < 60 ml/min and the reticulocyte count is inappropriate for the degree of anemia, consider anemia of chronic kidney disease If the reticulocyte count is elevated, consider hemolytic anemia
MCV is elevated (> 100 fL) See macrocytosis below

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• HEMOGLOBIN

• Overview
• Hemoglobin is a RBC protein that binds oxygen in the lungs for transport to tissues. Hemoglobin molecules contain 4 chains, and each chain is comprised of a heme molecule (iron + protoporphyrin IX) and a polypeptide called a globulin. Under normal conditions, there are four main types of chains - alpha chains, beta chains, gamma chains, and delta chains.

• Normal hemoglobin types
• Hemoglobin A (Adult hemoglobin) - Hemoglobin A is the most common form of hemoglobin in adults. It is made up of 2 alpha chains and 2 beta chains.
• Hemoglobin A₂ - Hemoglobin A₂ is a variant of hemoglobin A that comprises 1.5 - 3% of hemoglobin in adults. It is made up of 2 alpha chains and 2 delta chains.
• Hemoglobin F (fetal hemoglobin) - Hemoglobin F replaces embryonic hemoglobin and is the main hemoglobin during the last 7 months of gestation. It is made up of 2 alpha chains and 2 gamma chains. After birth, hemoglobin F is almost completely replaced by hemoglobin A. After 6 months of age, hemoglobin F makes up about 0.5% of hemoglobin. Hemoglobin F binds oxygen more tightly than adult hemoglobin to ensure proper intrauterine oxygen delivery.
• Hemoglobin E (embryonic hemoglobin) - Hemoglobin E is present during gestation from week 3 to 3 months. It is made up of 2 alpha chains and 2 epsilon chains. Hemoglobin E binds oxygen more tightly than adult hemoglobin to ensure proper intrauterine oxygen delivery.

• Abnormal hemoglobin types
• Bart's hemoglobin - Bart's hemoglobin is an abnormal form of hemoglobin that is composed of 4 gamma chains. Bart's hemoglobin occurs in homozygous alpha thalassemia. See alpha thalassemia below.
• Hemoglobin C - Hemoglobin C is hemoglobin A with a lysine substituted for glutamic acid at position 6 of each beta chain. See hemoglobin C disease below.
• Hemoglobin H - Hemoglobin H is a tetramer of 4 beta chains that forms in heterozygous types of alpha thalassemia. See alpha thalassemia below.
• Hemoglobin S - Hemoglobin S is hemoglobin A with a valine substituted for a glutamic acid at position 6 of each beta chain. Hemoglobin S causes sickle cell disease. See sickle cell disease below.

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• Alpha thalassemia
• Normal adult hemoglobin is composed of 2 alpha chains and 2 beta chains
• Alpha thalassemia is an inherited condition where genetic defects decrease the production of alpha chains. Alpha chains are encoded in two regions on each chromosome 16 giving them a total of 4 genes. Deletions or mutations of these genes can inhibit alpha chain production. When inadequate amounts of alpha chains are present, beta chains will combine with themselves to form tetramers called hemoglobin H. Hemoglobin H cannot transport oxygen and it will form inclusion bodies in RBCs that can lead to hemolysis. If no alpha chains are produced, hemoglobin Bart's (4 gamma chains) will form and in utero death will occur secondary to hydrops fetalis, a condition marked by diffuse tissue edema (see abnormal hemoglobin types and hemoglobin table above).
• The clinical course of alpha thalassemia is dependent on the number of genes affected and the amount of hemoglobin H produced (see table below). Long-term effects of alpha thalassemia include anemia from chronic hemolysis, hypersplenism, extramedullary hematopoiesis, iron overload with organ deposition and damage, hypercoagulability, and cholelithiasis.
• When necessary, transfusions are used to treat alpha thalassemia. Transfusions provide normal erythrocytes which suppress the drivers of abnormal hemoglobin production and help to prevent disease sequelae.
• In general, most patients who have α-thalassemia intermedia and produce hemoglobin H are clinically well and do not require transfusions

• Beta thalassemia
• Pathology
• Normal adult hemoglobin is composed of 2 alpha chains and 2 beta chains. Beta thalassemia is an inherited condition where genetic defects decrease the production of beta chains. Beta chains are encoded in one region on each chromosome 11 giving them a total of 2 genes. Deletions or mutations of these genes can inhibit beta chain production. When inadequate amounts of beta chains are present, alpha chains form tetramers that accumulate in RBCs and lead to premature cell death and ineffective erythropoiesis. Hemoglobin A is decreased, hemoglobin F is increased, and chronic anemia develops (see abnormal hemoglobin types and hemoglobin table above).
• Clinical course
• The clinical course of beta thalassemia is dependent on the severity of beta chain loss (see table below). Long-term effects of beta thalassemia include chronic transfusion-dependent anemia, hypersplenism, extramedullary hematopoiesis, iron overload from chronic transfusions causing organ deposition and damage, hypercoagulability, and cholelithiasis.
• Treatment
• Transfusions are used to treat significant beta thalassemia. Transfusions provide normal erythrocytes, which suppress the drivers of abnormal hemoglobin production and help to prevent disease sequelae.
• Stem cell transplant is an option in some patients and is potentially curative. Best outcomes are seen in patients younger than 14 years with an HLA-identical donor.
• A study published in 2022 described the use of gene therapy to treat 23 patients with transfusion-dependent beta thalassemia and a non–β^o/β^o genotype. Autologous stem cells were transduced with a virus that changed the beta globulin gene so that the cell produced functional beta globulin. After a median follow-up of 29.5 months, transfusion independence occurred in 91% of patients. [PMID 34891223] In 2022, the treatment received FDA approval under the trade name Zynteglo.
• In 2019, the FDA approved the first therapy to treat beta thalassemia, an injectable protein called Reblozyl® (luspatercept–aamt). In beta thalassemia, certain ligands inhibit the late stages of erythropoiesis. Reblozyl® binds these ligands and indirectly promotes RBC maturation. In a placebo-controlled trial, Luspatercept significantly reduced transfusion burden in patients with transfusion-dependent beta thalassemia. [PMID 32212518] In another study, it raised the average hemoglobin level in non–transfusion-dependent beta thalassemia. [PMID 36007538]
• In 2020, a report was published in the NEJM that described a case where a patient with transfusion-dependent beta-thalassemia was successfully treated with clustered regularly interspaced short palindromic repeats (CRISPR) gene editing. BCL11A is a transcription factor that represses gamma globin and fetal hemoglobin production in erythroid cells. CRISPR editing was used to knockout the gene that codes for BCL11A, and fetal hemoglobin levels increased. [PMID 33283989]

• Hemoglobin C disease
• Hemoglobin C disease is a genetically inherited hemoglobinopathy that occurs when glutamic acid is replaced with a lysine at position six on the beta chain (see abnormal hemoglobin types)
• If only one beta globin gene is affected, the patient will have hemoglobin C trait. Patients with hemoglobin C trait produce 55 - 65% hemoglobin A with 30 - 40% hemoglobin C and are typically asymptomatic. Peripheral blood smears may show target cells and intracellular crystals.
• If both beta globin genes are affected, hemoglobin C disease will occur. Patients with hemoglobin C disease produce no hemoglobin A and > 90% hemoglobin C (see hemoglobin table above). Hemoglobin C may crystallize causing increased RBC rigidity and shortened RBC lifespan, but it does not polymerize and cause sickling like hemoglobin S. Most patients with hemoglobin C disease are asymptomatic but they may have mild chronic hemolysis, splenomegaly, and anemia. Peripheral blood smears will show microcytosis, target cells, spherocytes, and crystallized hemoglobin.
• Hemoglobin C is often inherited with other hemoglobinopathies and it can affect the pathology of these conditions. For example, coinheritance of hemoglobin S and hemoglobin C tends to have a milder course than homozygous hemoglobin S (sickle cell disease).
• Hemoglobin C appears to protect against malaria, and therefore, it is primarily found in people of African descent. In the U.S., 1- 3% of blacks have hemoglobin C trait and <0.10% have hemoglobin C disease. Hemoglobin C detection is part of the standard newborn screening test.
• Treatment of hemoglobin C disease is generally not needed. If chronic hemolytic anemia is present, folic acid supplementation of 1 mg/day may be recommended to support high RBC turnover. Gallstones may also develop. No physical activity restrictions are required. [17]

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• SICKLE CELL DISEASE

• Epidemiology
• It's estimated that 300,000 newborns are born with sickle cell disease each year, with Sub-Saharan Africa, particularly Nigeria and the Congo, and India having the highest prevalence. In the U.S., about 100,000 people have the disease.
• In developed countries, treatment has improved substantially, with childhood mortality now similar to the general population and overall life expectancy reaching into the sixties. In poorer countries with limited resources, most children with sickle cell disease die before the age of five.

• Pathology
• Sickle cell disease is caused by hemoglobin S, an abnormal variant marked by the substitution of glutamic acid with valine at the sixth position of the hemoglobin A beta chain. When hemoglobin S is exposed to hypoxia, it elongates, causing the RBC to take on the shape of a sickle. Sickled RBCs do not pass freely through capillaries, leading to clumping and vessel occlusion with subsequent tissue hypoxia and pain (sickle cell crisis). Sickled cells are also fragile and prone to rupture, causing anemia.

• Sickle cell trait (SCT)
• Patients with two hemoglobin S genes will have sickle cell disease, while those with one hemoglobin S gene and one normal gene will have sickle cell trait. SCT is highly protective against severe malaria, and its prevalence ranges from 10 - 40% in certain parts of Africa and India. In the U.S., around 8% of African Americans are affected.
• Patients with sickle cell trait produce 35 - 45% hemoglobin S, and the rest is normal. The normal hemoglobin prevents the cells from sickling, so vaso-occlusive crises are not seen in SCT.
• The health consequences of sickle cell trait are the subject of much debate. Data from observational studies and case reports have suggested an increased risk for certain conditions (see below), and a high-profile lawsuit over a student-athlete death led the NCAA to enact universal sickle cell trait screening among all athletes. Possible risks of sickle cell trait include the following:
• Exercise-induced injuries - in observational studies, sickle cell trait has been associated with sudden death, exertional rhabdomyolysis, and other heat-related conditions. The incidence of these events is very low, and causality is unknown.
• Kidney disease - in observational studies, the prevalence of hematuria and chronic kidney disease has been higher in SCT patients
• Venous thromboembolism - observational studies have found an increased risk of venous thromboembolism among SCT patients
• Pregnancy complications - a small number of observational studies have found an increased risk of adverse pregnancy outcomes in SCT patients
• Renal medullary carcinoma - patients with SCT are at increased risk of renal medullary carcinoma, a rare cancer with an incidence of 1/20,000 to 1/39,000 among SCT patients [12,13,14,61]

• Effect of other hemoglobinopathies on severity of sickle cell disease
• Patients who are homozygous for hemoglobin S will only produce beta chains with the sickle cell defect. Other inherited hemoglobinopathies often occur with sickle cell disease, and these abnormalities can affect the severity of sickle cell disease.
• Alpha thalassemia reduces the amount of alpha chains produced. Coinheritance of alpha thalassemia and sickle cell disease tends to produce less severe disease because the thalassemia reduces the amount of hemoglobin in RBCs and this makes them less prone to sickling.
• Genetic variants that increase levels of hemoglobin F also reduce the severity of sickle cell disease because the hemoglobin F helps to prevent sickling
• Coinheritance of hemoglobin C disease leads to a milder disease course because hemoglobin C does not cause sickling
• Coinheritance of β-thalassemia intermedia allows for the production of some hemoglobin A and the disease can be milder. β-thalassemia major does not allow for the production of hemoglobin A and the disease course is similar to sickle cell disease
• See hemoglobin table for more [12,13,14]

• Sequelae of sickle cell disease
• The intermittent vaso-occlusive crises that occur in sickle cell disease lead to a number of long-term clinical sequelae
• Some of the more clinically relevant complications are listed below


• Functional asplenia - patients with sickle cell disease develop functional asplenia around the age of one year. Asplenia increases the risk for infections particularly from encapsulated bacteria like pneumococcus and haemophilus. In undeveloped nations, infections are a major cause of mortality in children with sickle cell disease. In developed nations, access to vaccines and prophylactic antibiotic therapy have greatly reduced the risk of death from childhood infections. See asplenic / hyposplenic patient care for more.
• Acute chest syndrome - acute chest syndrome is defined as a new pulmonary infiltrate accompanied by fever, chest pain, and shortness of breath. It is a leading cause of death among adults with sickle cell disease. Treatment includes antibiotics, respiratory support, and transfusions.
• Stroke - children with sickle cell disease are at a much greater risk for stroke with 11% of untreated patients experiencing a stroke by the age of 20 years. Transcranial doppler ultrasounds (TCD) are recommended from the ages of 2 to 16 years to help identify children at high risk. TCDs measure flow velocities in cranial arteries with higher velocities indicating stenosis and greater stroke risk. Velocities > 200 cm/sec are associated with an annual stroke risk of 10% and treatment is indicated. Velocities of 170 - 200 cm/sec are considered conditional and shorter follow-up is recommended. Normal scans are repeated annually. Treatment involves transfusions to maintain the hemoglobin S level at < 30% and has been shown to decrease stroke risk by > 90%.
• End-organ damage - over time, the repeated cycles of infarction and reperfusion begin to take their toll on bodily organs. Organ dysfunction and failure typically start to appear after the age of thirty, and any organ system can be affected. The table below gives some commonly affected organs and their sequelae

• Treatment
• Transfusions - transfusions are the primary treatment for sickle cell disease. They are used acutely to treat acute chest syndrome, stroke, and anemia. Preventively, transfusions are primarily used preoperatively and for stroke prevention (see stroke above). In general, the goal of transfusion therapy is a hemoglobin level that does not exceed 10 g/dl and a hemoglobin S level of < 30%. Chronic transfusions can have adverse effects including alloimmunization leading to hemolytic reactions, iron overload, and transmission of blood-borne pathogens.

• Hydroxyurea - hydroxyurea is an antineoplastic agent that inhibits ribonucleotide reductase. In sickle cell anemia, hydroxyurea increases the production of hemoglobin F by shifting RBC production from rapidly dividing precursor cells to progenitor cells that contain more hemoglobin F. This effect has made it a mainstay in the treatment of sickle cell disease for many years. Hydroxyurea is typically started at a dose of 15 mg/kg/day. It can then be titrated to a maximum dose of 35 mg/kg/day. After 6 months of use, hemoglobin F levels typically double. Hydroxyurea is myelosuppressive and blood counts must be monitored closely during therapy. Hydroxyurea (Hydrea®) has been around for a long time, and it has a cheap generic. Two newer versions of hydroxyurea, Siklos® and Droxia®, are also available, but they are more expensive. See the Droxia PI for hydroxyurea dosing information in sickle cell disease. [23]

• Infection prevention - functional asplenia and a number of other factors greatly increase the risk of infection in patients with sickle cell disease. All patients with sickle cell disease should have recommended immunizations for asplenic patients (see immunizations in asplenia). Antibiotic prophylaxis with a penicillin is recommended in all children until the age of five years (see antibiotic prophylaxis in asplenia).

• Stem cell transplantation - stem cell transplantation is the only curative treatment for sickle cell disease with a success rate approaching 90% when marrow or cord blood stem cells from HLA-matched siblings are used. Unfortunately, only 10 - 20% of sickle cell patients have unaffected matched sibling donors. [12,13]

• Gene therapy - gene therapy is a rapidly evolving field of medicine, and it is being studied in sickle cell disease as a possible treatment. A study published in 2022 described the use of gene therapy to treat 35 patients with sickle cell disease and at least 4 severe vaso-occlusive events in the prior 24 months. Autologous stem cells were transduced with a virus that changed the beta globulin gene so that the cell produced an antisickling hemoglobin. After a median follow-up of 17.3 months, median hemoglobin levels increased from 8.5 g/dl to 11 g/dl, and there were no severe vaso-occlusive events. [PMID 34898139] Another paper published in 2021 described the use of gene therapy to alter the BCL11A gene. BCL11A is a transcription factor that represses gamma globin and fetal hemoglobin production in erythroid cells. Using gene therapy, BCL11A production was suppressed in 6 patients with sickle cell disease, and fetal hemoglobin levels increased. [PMID 33283990]

• CRISPR gene editing - In 2020, a report was published in the NEJM that described a case where a patient with sickle cell disease was successfully treated with clustered regularly interspaced short palindromic repeats (CRISPR) gene editing. CRISPR gene editing was used to knock out the gene that codes for BCL11A, a transcription factor that represses gamma globin and fetal hemoglobin production in erythroid cells. After treatment, the patient's fetal hemoglobin levels increased. [PMID 33283989]

• Adakveo® (crizanlizumab-tmca) - in 2019, the FDA approved Adakveo® for the prevention of vaso-occlusive crises in patients with sickle cell disease. In vaso-occlusive crises, a molecule called P-selectin is expressed on the surface of endothelial cells. P-selectin promotes erythrocyte adhesion which helps propagate vaso-occlusion. Adakveo® is an antibody to P-selectin that blocks the interaction between P-selectin and erythrocytes. In a study involving 198 patients, Adakveo® was shown to almost half the number of pain crises per year. [PMID 27959701] [Adakveo PI]

• Oxbryta® (voxelotor) - Oxbryta® (voxelotor) has been approved by the FDA to treat sickle cell disease in patients ≥ 4 years old. Voxelotor is an oral hemoglobin S polymerization inhibitor that reversibly binds to hemoglobin and increases its affinity for oxygen, thus preventing the sickled state. In a study involving 274 patients, hemoglobin levels increased by > 1 g/dl over 24 weeks in 51% of voxelotor-treated patients compared to 7% of placebo-treated patients. [PMID 27959701] [Oxbryta PI]

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• MICROCYTIC ANEMIA | Iron-deficiency anemia

• Overview
• Iron-deficiency anemia is the number one cause of anemia worldwide affecting about 2 billion people

• Pathology
• Iron homeostasis is tightly controlled in humans. Total body iron amounts to about 4 - 5 grams with 65% of iron found in hemoglobin, 15 - 30% as ferritin, 4% in myoglobin, and 0.1% as transferrin. Under normal conditions, the majority of body iron is recycled from dying RBCs by macrophages and about 1 - 2 mg per day is absorbed from food in the intestine. Men lose an average of 0.6 mg/day of iron in the feces and women lose an average of 1.3 mg/day due to fecal loss and menses.
• Because high iron levels are toxic to the body, the hormone hepcidin helps to control iron levels by suppressing iron release from the liver and inhibiting iron absorption from the intestines. High iron levels in the blood and liver stimulate hepcidin release while low iron levels and tissue hypoxia suppress hepcidin release. Hepcidin release is also stimulated by inflammatory mediators, and prolonged inflammation can lead to decreased iron levels and anemia of chronic inflammation.
• Iron-deficiency anemia occurs when iron stores are unable to maintain normal erythropoiesis. The iron deficient state typically occurs through excessive blood loss (e.g. menses, gastrointestinal bleeding), inadequate intake or increased demand (e.g. vegetarian diet, pregnancy) or impaired absorption (e.g. celiac disease, bariatric surgery).

• Diagnosis
• Iron-deficiency anemia is diagnosed with iron studies. The best tests for assessing iron status are the serum ferritin and the transferrin saturation.
• A serum ferritin < 30 mcg/L and/or a transferrin saturation < 16% is inadequate to support erythropoiesis, and these findings are highly suggestive of iron-deficiency anemia
• Ferritin is also an acute phase reactant and may be elevated in chronic inflammatory states. The total iron binding capacity (TIBC) may be helpful in distinguishing iron-deficiency anemia from anemia of chronic inflammation. A high TIBC is consistent with iron deficiency, and a low TIBC is consistent with anemia of inflammation. If iron-deficiency anemia and anemia of inflammation are both present, the TIBC is not helpful.

• Treatment
• The underlying cause of iron deficiency (see risk factors) should be addressed. Oral iron replacement is preferred in most cases, while intravenous iron may be appropriate in others (e.g. impaired absorption, rapid correction desired).

• Screening infants and children
• The CDC recommends screening the following infants and children for iron deficiency at age 9 -12 month and 6 months later:
• Preterm or low-birthweight infants
• Infants fed a diet of non-iron-fortified infant formula for greater than 2 months
• Infants introduced to cow's milk before age 12 months
• Breast-fed infants who do not consume a diet adequate in iron after age 6 months (i.e., who receive insufficient iron from supplementary foods)
• Children who consume greater than 24 oz daily of cow's milk
• Children who have special healthcare needs (e.g., children who use medications that interfere with iron absorption and children who have chronic infection, inflammatory disorders, restricted diets, or extensive blood loss from a wound, an accident, or surgery) [4]
• USPSTF
• The USPSTF states that the current evidence is insufficient to assess the balance of benefits and harms of screening for iron deficiency anemia in children ages 6 to 24 months [8]

• Preventing iron deficiency in pregnancy
• CDC recommendations
• The CDC recommends 30 mg a day of supplemental elemental iron in order to prevent iron deficiency in pregnancy
• Women should start supplementation as soon as they find out they are pregnant [4]
• USPSTF
• The USPSTF states that the current evidence is insufficient to assess the balance of benefits and harms of screening for iron deficiency anemia in pregnant women to prevent adverse maternal health and birth outcomes
• The USPSTF states that the current evidence is insufficient to assess the balance of benefits and harms of routine iron supplementation for pregnant women to prevent adverse maternal health and birth outcomes [8]

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• MICROCYTIC ANEMIA

• Lead poisoning
• Lead poisoning is an important cause of microcytic anemia, particularly in children. Lead induces microcytosis and anemia through several mechanisms, including inhibition of heme synthesis by blocking the insertion of iron into protoporphyrin, decreased RBC lifespan, and suppression of erythropoietin production by the kidneys. Chronic exposure to lead can lead to developmental delay in children and cardiovascular disease in adults.
• The average lead level in U.S. adults is 0.92 mcg/dl. Levels > 3.5 mcg/dl are considered elevated in children, while levels > 5 mcg/dl are elevated in adults. Levels ≥ 10 mcg/dl require monitoring and investigation. Chelation therapy is recommended for levels > 45 mcg/dl in children and > 80 mcg/dl in adults. Lead-induced anemia is seen at levels > 50 mcg/dl.
• Lead is absorbed in the digestive tract or lungs when ingested or inhaled. Children absorb about 50% of the lead they ingest, whereas adults only absorb about 10%. Concomitant iron deficiency increases absorption. Once absorbed, lead is distributed between the soft tissues, bones, and teeth. In adults, 80 - 95% of lead is stored in the bones, whereas in children, only about 70% stays in the bones, making them more susceptible to its toxic effects. Lead is slowly eliminated from the body through the urine and bile.
• If lead poisoning is confirmed, a search for the source should ensue. See sources of lead from the CDC for more. After removal of the offending agent, lead levels begin to decline slowly. A study that measured the amount of time it took for toxic levels to decline in children < 6 years of age is detailed in the table below. [27,28,59]

• Reference [29]

Time for lead levels to fall below 10 mcg/dl in children < 6 years of age
Initial lead level (mcg/dl)	Median days to fall below 10 mcg/dl
10 - 14 (N=452)	237
15 - 19 (N=315)	424
20 - 24 (N=112)	659
25 - 29 (N=59)	954
≥ 30 (N=59)	1083

• Thalassemias
• Worldwide, the thalassemias are a common cause of microcytic anemia. See alpha thalassemia and beta thalassemia above.

• Rare causes of microcytic anemia
• Hypotransferrinemia - Hypotransferrinemia is caused by defects in the gene that codes for transferrin which lead to transferrin deficiency and inadequate iron availability. It presents as microcytic and hypochromic anemia early in life with low serum iron and high ferritin levels. Transferrin levels are undetectable or below normal and fully saturated. It can be treated with apotransferrin transfusions. [31]

• Iron-refractory iron deficiency anemia (IRIDA) - IRIDA is caused by defects in the TMPRSS6 gene that codes for matriptase-2, a protein that senses iron deficiency and blocks hepcidin transcription. In IRIDA, hepcidin levels are inappropriately elevated and iron absorption is inhibited. IRIDA presents with microcytic anemia, low TSAT, and normal or reduced ferritin that does not have an expected response to oral or IV iron. [31]

• Sideroblastic anemia - sideroblastic anemia is a microcytic anemia that is caused by abnormalities in heme synthesis which can be inherited through genetic defects or acquired. Abnormal heme synthesis leads to the deposition of iron in the mitochondria of erythroid precursor cells. Iron-laden mitochondria form a ring around the nucleus of the cell giving it the characteristic "ringed sideroblast" appearance which can be seen in the bone marrow (sideroblast illustration). Inherited forms of sideroblastic anemia arise from defects in the genes that code for enzymes involved in heme synthesis, iron-sulfur cluster biogenesis, and mitochondrial metabolism. Acquired forms of sideroblastic anemia can arise from drugs (e.g. isoniazid, chemotherapy), toxins (e.g. lead), copper deficiency, and chronic myelodysplastic diseases. [32]

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• NORMOCYTIC ANEMIA | Anemia of inflammation

• Overview
• Anemia of inflammation (AI), also called anemia of chronic disease, is the second most common cause of anemia. It is estimated that 40% of all anemias are due to AI or have a component of AI which means it affects more than 1 billion people worldwide.
• AI by itself is a normocytic normochromic anemia, but it often occurs with iron-deficiency anemia, so microcytosis and hypochromia can also be present
• Unlike iron-deficiency anemia where there is an inadequate supply of body iron, iron stores in AI are typically sufficient, but the iron is unable to distribute appropriately to maintain normal erythropoiesis. The reason for this is multifactorial and not completely understood.
• Since AI and iron-deficiency anemia can coexist, and there is no definitive test for AI, differentiating between the two can be diagnostically challenging [24,25]

• Pathological processes through which AI is thought to occur
• Disruption of iron homeostasis - in AI, inflammatory mediators (e.g.interleukin-6) alter iron homeostasis by stimulating hepcidin production which in turn causes a decrease in the intestinal absorption of iron and inhibits the release of iron from macrophages. Iron retention by macrophages is particularly disruptive since macrophages supply > 90% of the iron needed for erythropoiesis.
• Suppression of erythropoiesis - in the bone marrow, inflammatory mediators (e.g. tumor necrosis factor alpha, interferon-γ) promote myelopoiesis and lymphopoiesis which in turn suppresses erythropoiesis. Inflammation also reduces the production of erythropoietin and blunts the response of marrow to its effects.
• Decrease in RBC lifespan - in the inflammatory state, RBC lifespan is decreased from an average of 120 days to around 90. This is thought to occur secondary to macrophage stimulation and "bystander" damage to erythrocytes from surrounding inflammatory processes (e.g. inadvertent complement and antibody deposition on RBCs). [24,25]

• Diseases associated with AI
• Cancer and hematological malignancies
• Infections
• Immune-mediated diseases (e.g. rheumatoid arthritis, lupus)
• Inflammatory diseases
• Chronic kidney disease
• Congestive heart failure
• Chronic pulmonary disease
• Obesity
• Anemia of the elderly
• Anemia of critical illness (accelerated course) [25]

• Diagnosis
• AI by itself presents is a normochromic, normocytic anemia with normal to high ferritin levels, reduced transferrin saturation, and reduced transferrin. Depending on the study, 20 - 85% of patients with AI also have a true iron deficiency. When both coexist, iron studies and RBC indices are less definitive, and further evaluation may be necessary.
• The table below details how different iron studies are affected by AI and iron-deficiency anemia

• References [24,25,26]

Findings in AI and iron-deficiency anemia
Ferritin Anemia of inflammation Ferritin is predominantly secreted by macrophages and hepatocytes. In AI, it is typically normal or elevated. Iron-deficiency anemia Ferritin < 30 mcg/ml - ferritin levels < 30 mcg/ml mean true iron deficiency is present because levels below 30 are inadequate to maintain normal erythropoiesis Ferritin 30 - 100 mcg/ml - ferritin levels in the 30 - 100 mcg/ml range can occur in both iron-deficiency anemia and AI. Ferritin is an acute phase reactant, so the low levels seen in iron deficiency may be offset by increased secretion from stimulated macrophages. Ferritin > 100 mcg/ml - ferritin levels > 100 mcg/ml are uncommon in true iron deficiency and suggest another disorder
Serum iron Anemia of inflammation Serum iron levels are low in AI because hepcidin suppresses iron release from macrophages and intestinal absorption Iron-deficiency anemia Iron levels are low due to an inadequate amount of iron stores
TIBC Anemia of inflammation TIBC is an indirect measure of transferrin concentration. Transferrin is secreted by the liver, and inflammatory mediators suppress its secretion. In AI, the TIBC is typically low. Iron-deficiency anemia In iron-deficiency anemia, transferrin release is stimulated, so the TIBC is typically high-normal or elevated
Transferrin saturation Anemia of inflammation Low serum iron levels in AI reduce transferrin saturation while inflammation reduces transferrin levels. Depending on the degree of these two effects, transferrin saturation may be low or normal. Iron-deficiency anemia Transferrin saturation < 20% - may occur in iron deficiency, AI, or a combination of the two Transferrin saturation > 20% - uncommon in iron deficiency
Soluble transferrin receptor Overview Transferrin receptor is a transmembrane protein that regulates iron uptake by cells. Cellular expression of transferrin receptor is increased when a cell needs iron. Soluble transferrin receptor is a cleavage product of transferrin receptor that can be measured in the blood. Erythroid progenitor cells are the primary cells that need iron, and 80% of soluble transferrin receptor in the blood comes from these cells. Inflammation does not affect soluble transferrin receptor levels, so this makes it a specific marker for iron deficiency. This lab is not widely available. Anemia of inflammation Levels are typically normal Iron-deficiency anemia Levels are increased

• Treatment
• When treating AI, it's important to consider that in some cases, the pathology behind AI plays an important role in the body's defense mechanism, particularly with infections and certain cancers. Circulating pathogens need iron to proliferate and certain solid tumors and hematologic malignancies express transferrin receptors. Suppressing iron levels can help to contain these conditions. Low iron levels also promote myelopoiesis and lymphopoiesis which provide the cells needed to combat these ailments.
• Curing the underlying disorder is the only way to resolve AI, but this is not always possible. When anemia becomes significant and treatment is required, iron supplementation and/or erythropoiesis-stimulating agents are the two main options. There are no good clinical trials to guide the use of these therapies in AI. In general, hemoglobin targets similar to those used in chronic kidney disease (11 - 12 g/dl) appear to be safe. See treatment of anemia in chronic kidney disease for more. [24,25,26]

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• NORMOCYTIC ANEMIA | Chronic kidney disease (CKD)

• Pathology
• The kidneys release erythropoietin in response to tissue hypoxia. Erythropoietin stimulates RBC production in the bone marrow. In CKD, erythropoietin release is reduced and RBC production is diminished. It's important to note that erythropoietin levels are often normal or slightly elevated in patients with CKD, but they are inappropriate for the anemia that is present with similarly anemic patients without CKD having erythropoietin levels 10 - 100 times that of patients with CKD. Because of this, erythropoietin levels are not recommended in the workup of CKD-induced anemia.
• Other factors that play a role in the anemia of CKD include shortened RBC lifespan, anemia of chronic inflammation, vitamin deficiencies, and retained toxins from uremia that suppress erythropoiesis
• Anemia from CKD can start to appear when the GFR falls below 60 ml/min. The table below gives the prevalence of anemia and average Hg stratified by GFR in a sample of 15,419 U.S. adults. [35,36,37]

• Data is from a sample of 15,419 noninstitutionalized U.S. adults
• Anemia was defined as Hg < 12 g/dl for men and < 11 g/dl for women
• Reference [33]

Anemia in CKD
GFR (ml/min)	% or patients with anemia	Average Hg (g/dl)
≥ 90	1.8%	14.2
60 - 89	1.3%	14.2
30 - 59	5.2%	13.6
15 - 29	44.1%	11.8

• Diagnosis
• There is no single measure that can definitively diagnose anemia of CKD. Measuring an erythropoietin level seems intuitive, but levels are often normal or slightly elevated in CKD and are noninformative. Further complicating matters is the fact that patients with CKD often have some degree of anemia of chronic inflammation which presents similarly. The table below gives the recommended testing from the National Kidney Foundation along with associated findings in CKD. [34,35]

• References [34,35]

Findings in Anemia of CKD
RBC indices Anemia of CKD is typically a normochromic, normocytic anemia
Reticulocyte indices Because erythropoietin levels are not a good marker of erythropoiesis in CKD, reticulocyte measures are recommended to assess erythropoietic activity Reticulocyte count (normal 0.5 - 2.5%) - in anemia of CKD, the reticulocyte count may be normal or slightly elevated, but it is inappropriately low for the presence of anemia Reticulocyte index - a reticulocyte index ≥ 2 is considered an appropriate response to anemia. An index < 2 is considered inappropriate and consistent with anemia of CKD. Reticulocyte production index - a reticulocyte production index > 3 is considered an appropriate response to anemia. An index ≤ 2 is considered inappropriate and consistent with anemia of CKD.
Iron studies Ferritin Ferritin < 30 ng/ml: ferritin levels < 30 ng/ml are inadequate for erythropoiesis and indicate a true deficiency of iron stores Ferritin 30 - 300 ng/ml: levels in this range are less informative because ferritin is an acute phase reactant and is often elevated in inflammatory conditions like CKD Ferritin > 300 ng/ml: most patients with CKD and ferritin levels > 300 ng/ml will have normal bone marrow iron stores Transferrin saturation (TSAT) - the TSAT is the most commonly used measure in CKD to assess iron availability for erythropoiesis. A TSAT < 20% in patients with CKD is indicative of inadequate iron availability for erythropoiesis. Reticulocyte hemoglobin content (CHr) - the CHr provides a more acute measure of iron availability for erythropoiesis than ferritin and TSAT. Levels < 28 pg in adults typically reflect inadequate iron availability. The CHr is not available at all labs.

• Treatment
• Anemia of CKD is treated with erythropoietin-stimulating agents (ESA) along with careful monitoring of iron indices to ensure that there is sufficient functional iron available for erythropoiesis. While ferritin levels > 100 ng/ml typically indicate adequate iron stores, studies have consistently found that iron supplementation at ferritin levels up to 1200 ng/ml will produce an increase in hemoglobin in CKD patients
• The table below gives the recommendations for treating anemia of CKD from the International Society of Nephrology and the American Kidney Foundation

• References [35,36]

Treatment Recommendations for Anemia of CKD in Adults
Hemoglobin Anemia in males: Hg < 13 g/dl Anemia in females: Hg < 12 g/dl Target hemoglobin range: 11 - 13 g/dl
Iron therapy CKD patients with anemia not at goal who are not receiving ESA therapy A trial of IV iron therapy may be beneficial if TSAT ≤ 30% and ferritin ≤ 500 ng/ml Patients who are not on dialysis may benefit from a 1 - 3 month trial of oral iron CKD patients with anemia not at goal who are receiving ESA therapy A trial of IV iron therapy may be beneficial if TSAT ≤ 30% and ferritin ≤ 500 ng/ml Patients who are not on dialysis may benefit from a 1 - 3 month trial of oral iron Check TSAT and ferritin every 3 months during ESA therapy
ESA therapy targets Non-dialysis patients Hg ≥ 10 g/dl: do not initiate ESA therapy Hg < 10 g/dl: consider ESA therapy on an individual basis. Considerations should include rate of fall of Hb concentration, prior response to iron therapy, the risk of needing a transfusion, the risks related to ESA therapy and the presence of symptoms attributable to anemia. Dialysis patients Start ESA therapy when Hg is 9 - 10 g/dl to prevent Hg < 9 g/dl. Some patients may benefit from starting therapy at Hg > 10 g/dl. In general, ESA therapy should be adjusted to maintain a Hg between 10 - 13 g/dl. Some individuals may have improvements in quality of life at the higher end of this range, but levels > 13 g/dl are not recommended. When initiating therapy, measure Hg levels at least monthly. Once a maintenance dose has been established, measure Hg at least every 3 months in non-dialysis patients and monthly in dialysis patients.
Erythropoietin-stimulating agents Epoetin alfa (Epogen®, Procrit®, Retacrit®) Epogen®, Procrit®, and Retacrit® are all epoetin alfa. Retacrit is a biosimilar for Epogen and Procrit. For adult CKD patients not on dialysis, the recommended starting dose is 50 - 100 units/kg by subcutaneous or intravenous route three times weekly. Comes in vial sizes that range from 2000 - 40,000 units per vial. [Epogen PI] Darbepoetin alfa (Aranesp®) Darbepoetin alfa is a long-acting erythropoietin analog. For adult CKD patients not on dialysis, the recommended starting dose is 0.45 mcg/kg by subcutaneous or intravenous route every 4 weeks. Comes in vials and prefilled syringes. Prefilled syringes come in doses that range from 10 - 500 mcg. [Aranesp PI] Methoxy polyethylene glycol-epoetin beta (Mircera®) Mircera is a long-acting erythropoietin analog. For adult CKD patients not on dialysis, the recommended starting dose is 1.2 mcg/kg by subcutaneous route every month. Comes in prefilled syringes in doses that range from 30 - 360 mcg. [Mircera PI] Daprodustat (Jesduvroq®) Daprodustat increases the transcription of erythropoietin genes by inhibiting hypoxia inducible factor (HIF), prolyl 4-hydroxylases(PH)1, PH2, and PH3. It is a once-daily tablet approved for use in adults with CKD-induced anemia who have been receiving dialysis for at least 4 months. [Jesduvroq PI]

• Hemolytic anemia
• Hemolytic anemia is often normocytic (e.g. autoimmune hemolytic anemia, mechanical hemolysis) but can also be microcytic (e.g. thalassemia, sickle cell disease). See hemolytic anemia for more.

• Hemorrhagic anemia
• Anemia may occur through blood loss. After an acute hemorrhage that is stopped, it typically takes 3 - 6 weeks for RBC concentrations to return to normal. In chronic blood loss, the absorption of iron often cannot meet the demands of RBC production and microcytic anemia may occur.

• Aplastic anemia
• Aplastic anemia occurs when bone marrow damage or destruction leads to decreased RBC production. Causes of aplastic anemia include chemotherapy, toxic chemicals (e.g. benzene), and autoimmune disorders (e.g. lupus). In half the cases of aplastic anemia, an etiology is never found.

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• HEMOLYTIC ANEMIA

• Epidemiology
• Hemolytic anemia accounts for about 5% of all anemias
• Acute autoimmune hemolytic anemia (AHA) is a rare condition with an incidence of only 1 - 3 cases per 100,000 people/year. Patients with certain underlying conditions are at much greater risk for AHA with up to 10% of systemic lupus erythematosus (SLE) patients, 5 - 10% of chronic lymphocytic leukemia patients, and 4.5% of hematopoietic stem cell transplant recipients being affected. [38]

• Types of hemolytic anemia
• There are 3 basic types of hemolytic anemia: autoimmune, mechanical hemolysis, and RBC abnormalities. All 3 types lead to the destruction of RBCs, but the underlying process is different for each type. It's also important to note that the pathology behind some causes of hemolytic anemia is not completely understood. RBC hemolysis can occur in the circulation (intravascular) or in tissues like the liver and spleen where macrophages reside (extravascular).
• The table below gives the underlying pathology and associated risk factors for each type of hemolytic anemia.

Autoimmune hemolytic anemia (AHA)
Pathology Autoimmune hemolytic anemia occurs when antibodies to RBC surface antigens bind RBCs and promote their destruction. Autoimmune hemolytic anemia is divided into warm, cold, and mixed types. Warm autoimmune hemolytic anemia (WAHA) WAHA is called "warm" because binding occurs at most temperatures but is optimal at 98.6° F. WAHA is the most common type of autoimmune hemolytic anemia accounting for 48 - 70% of cases. Antibodies are typically of the IgG type, but can also be IgA. In WAHA, antibody-coated RBCs are typically destroyed in the liver or spleen by macrophages. Warm IgG antibodies do not normally cause RBC agglutination. Cold autoimmune hemolytic anemia (CAHA) CAHA is less common than WAHA, accounting for 15 - 25% of AHA cases. Cold agglutinins are IgM antibodies that bind the I/i antigen on RBCs. They are most reactive between 39 and 70° F, hence the name "cold." If their concentration is high enough, they can cause RBC agglutination and fixation of C3 to the RBC membrane. C3-tagged RBCs may undergo intravascular hemolysis if the complement system is completed, but more commonly, they are removed by macrophages in the spleen and liver. Cold agglutinin antibodies are present at low titers in almost all humans, and they only become clinically significant at higher titers. Cold agglutinins may form secondary to an underlying condition (e.g. cancer, Mycoplasma pneumonia, mononucleosis), in which case, a person is said to have "cold agglutinin syndrome." In rarer cases, cold agglutinins form from a clonal low-grade B-cell lymphoproliferative disorder, and affected individuals are said to have "cold agglutinin disease." Patients with cold agglutinin disease may develop acrocyanosis (bluish or purple toes or fingers) when exposed to cold temperatures. They also have chronic transfusion-dependent anemia and are at much greater risk for thromboembolism. [58] Mixed autoimmune hemolytic anemia Mixed AHA is characterized by the presence of both warm (IgG) and cold (IgM) antibodies. Mixed AHA accounts for about 8 - 10% of AHA cases, and it is typically severe with significant anemia. Risk factors for AHA Autoimmune diseases - SLE (up to 10% of patients), ITP, Rheumatoid arthritis, Sjogren's syndrome, Myasthenia gravis, Autoimmune hepatitis, Primary biliary cirrhosis, Ulcerative colitis, Sarcoidosis, Eosinophilic fasciitis, Pernicious anemia, Thyroiditis Hematologic disorders - leukemias, lymphomas, myelodysplastic syndromes, immunodeficiencies Infections Viruses: HIV, Hepatitis C, Hepatitis B, Cytomegalovirus, Parvovirus B19 Bacteria: tuberculosis, brucellosis, babesiosis Mycoplasma pneumonia and Epstein Barr virus: both have antigens that crossreact with the I antigen (Mycoplasma) and i antigen (Epstein Barr) on RBCs; common cause of elevated cold agglutinin titers, rarely lead to symptomatic hemolysis Drugs Antibiotics: ceftriaxone, piperacillin NSAIDs: diclofenac Cancer therapy: oxaliplatin, nivolumab Other - Allogeneic hematopoietic stem cell transplant recipients (up to 4.5% of patients over 3 years) [38,40]
Mechanical hemolysis
Pathology Mechanical hemolysis of RBCs occurs when abnormal physical elements within the circulation break apart RBCs through direct trauma. RBCs may be hemolyzed entirely or broken into fragments called schistocytes that are visible on a peripheral smear. The mechanism of the destruction depends upon the underlying condition. Below are some of the more common causes of mechanical hemolysis. Artificial heart valves Subclinical hemolysis has been detected in up to 51% of mechanical valve recipients, 10% of bioprosthetic valve recipients, and 15% of transcatheter valve replacement recipients. Artificial heart valves are thought to damage RBCs through increased shear stress. Increased shear stress may occur from displaced material (e.g. a loose suture), abnormal flow through the valve (more common with ball-and-cage valves), valve failure/degeneration, and the most common cause, paravalvular leak. Paravalvular leak occurs when there is an incomplete seal between the valve and the adjacent heart tissue which allows regurgitant blood flow through the defect when pressure rises. Paravalvular leaks are seen in up to 17% of mitral valve replacements and 10% of aortic valve replacements. Most paravalvular leaks are subclinical, and symptomatic hemolysis is seen in < 1% of patients with modern heart valves. Spontaneous resolution of hemolysis is common with aortic valves (75%) but less so with mitral valves (15%). Patients with refractory hemolytic anemia or heart failure may require surgical valve repair. [42, 56] Thrombotic thrombocytopenic purpura (TTP) Incidence - Overall, TTP is a very rare condition with an annual incidence of 2.9 cases per 1 million adults and 0.1 cases per 1 million children. Pathology - TTP is a thrombotic disorder caused by a deficiency in an enzyme called ADAMTS13, which cleaves von Willebrand factor multimers into smaller pieces, reducing their ability to form clots (see von Willebrand disease for more). Reduced ADAMTS13 activity promotes clot formation in small blood vessels, and RBCs are hemolyzed when they travel through the damaged vessels. Deficiencies in ADAMTS13 can be inherited from abnormal ADAMTS13 genes or acquired through the development of anti-ADAMTS13 antibodies (more common). Risk factors - Risk factors for acquired TTP include pregnancy, cancer, HIV, lupus, infections, and medications (e.g. chemotherapy, ticlopidine, clopidogrel, cyclosporine, alemtuzumab, quinine). Most cases occur in young or middle adulthood, with Blacks being affected seven times more than other races and women more than men. Symptoms - Symptoms of TTP are related to hemolysis and microthrombosis. In a registry of 78 TTP patients, presenting symptoms included gastrointestinal complaints (69%), bleeding, purpura, or hematuria (54%), and severe neurologic abnormalities (41%). Significant kidney dysfunction is rare (5% of patients) and suggests an alternative diagnosis, such as hemolytic-uremic syndrome. Diagnosis - ADAMTS13 activity levels, which are normally greater than 50%, are used to diagnose TTP. A level less than 10% is diagnostic for TTP, while a level greater than 20% makes it unlikely. Because levels can take days to come back, and TTP requires immediate treatment, clinical probability tools have been developed to help identify possible cases while labs are pending. The PLASMIC Score for TTP uses cancer and transplant status, along with some common lab values, to assign high, intermediate, or low TTP probability. It's recommended that patients with intermediate and high probability scores receive immediate plasmapheresis and corticosteroids (see PLASMIC Score calculator) Treatment - TTP is treated with immunosuppressants (e.g. glucocorticoids, rituximab) and plasma exchange, which replaces the deficient ADAMTS13 enzyme and removes inhibitory antibodies. With treatment, survival is greater than 80% after an initial episode, followed by a relapse rate of about 41% over 7.5 years. A drug called caplacizumab (Cablivi®) was FDA-approved in 2019 to treat TTP. Caplacizumab is an antibody that inhibits the interaction between von Willebrand factor and platelets. [Cablivi® PI] [43,60,62] Hemolytic uremic syndrome (HUS) Pathology - HUS is caused by small vessel endothelial cell damage that promotes thrombi formation. RBCs are hemolyzed in the damaged vessels, and the renal microcirculation is occluded, leading to kidney failure. There are two types of HUS: Shiga-like toxin-associated HUS and Atypical HUS. Shiga-like toxin-associated HUS (Stx-HUS) - Shiga-like toxin-associated HUS (90% of HUS cases) is caused by toxins released from bacteria, primarily Shigella dysenteriae and E. Coli. Stx-HUS typically presents with severe abdominal pain and bloody diarrhea, and it is most common in children under 5 years of age, with an incidence of 6.1 cases per 100,000/year. Atypical HUS - Atypical HUS (5 - 10% of HUS cases) is not entirely understood but is believed to be caused by abnormal complement system activation either through genetic mutations in complement components or the formation of autoantibodies to alternative-pathway complement-regulating factors. Risk factors for atypical HUS, which has an annual incidence of 2 per 100,000, include Streptococcus pneumoniae infection, viruses, drugs, cancer, transplantation, and genetic predisposition. Treatment - Treatment of Stx-associated HUS is generally supportive, and most patients make a full recovery. Atypical HUS is treated with complement system inhibitors like eculizumab and ravulizumab. [43,44,62] Disseminated intravascular coagulation (DIC) Pathology - DIC is a condition marked by inappropriate activation of coagulation pathways, leading to clotting factor consumption, microvascular thrombosis, hemorrhaging, excessive fibrin formation, and multiple organ failure. RBCs traveling through damaged and thrombosed vessels are hemolyzed. Risk factors - DIC can be an acute process (most common) or a chronic condition. The main risk factors for DIC are sepsis, cancer, pregnancy complications, and trauma. Diagnosis - a scoring system that incorporates the platelet count, D-dimer value, prothrombin time, and fibrinogen level has been developed that can be used to estimate the probability of DIC. See ISTH criteria for DIC calculator Treatment - The primary treatment for DIC is to correct the underlying disorder. Significant bleeding is treated with platelet and clotting factor replacement (fresh frozen plasma). If bleeding is not life-threatening, heparin may be used to prevent thrombosis. [45,62]
RBC abnormalities
Pathology RBC abnormalities are typically inherited disorders that cause RBCs to take on an abnormal shape. Abnormal RBCs are destroyed by immune cells in the spleen, liver, and bone marrow. Paroxysmal nocturnal hemoglobinuria (PNH) PNH is a rare disorder caused by a genetic defect that affects the proteins on the surface of RBCs. In PNH, RBC membranes lack two proteins, CD59 and CD55, that help regulate and inhibit the complement system; this makes the cells much more susceptible to complement fixation and hemolysis. The lack of CD59 leads to intravascular hemolysis from the C5-C9 membrane-attack complex, and the lack of CD55 causes C3 opsonization and extravascular hemolysis in the liver and spleen. The name of the disease is derived from the fact that the morning urine is often dark from bilirubin, and originally, it was thought that hemolysis mostly occurred at night. It has since been shown that hemolysis occurs throughout the day and is chronic. Sequelae of PNH include large vessel thrombosis, aplastic anemia, and renal failure from bilirubin overload. Two antibodies that bind and inhibit complement C5 have been approved to treat PNH. The first one was approved in 2007, and it is called eculizumab (Soliris). The second one was approved in 2021, and it is named ravulizumab (Ultomiris). Another drug called pegcetacoplan that inhibits complement C3 was found to be more effective than eculizumab in a head-to-head trial. [PMID 33730455]. In 2021, pegcetacoplan was FDA-approved to treat PNH, and it is marketed under the name Empaveli. [41,56] Glucose-6-phosphate dehydrogenase (G6PD) deficiency Pathology - G6PD is an enzyme that helps prevent oxidative damage in RBCs. When G6PD is deficient, oxidative stresses cause hemoglobin to denature and the RBC becomes susceptible to hemolysis. Denatured hemoglobin forms inclusion bodies called Heinz bodies inside of RBCs that are removed by splenic macrophages. When macrophages remove Heinz bodies, they cause the RBC to take on an irregular shape referred to as a "bite cell." Hemolysis in G6PD deficiency can be intravascular, but is mostly extravascular. Treatment of G6PD deficiency is supportive, and it typically resolves once the inciting factor is removed. Prevalence - G6PD deficiency is protective against severe malaria, so it is mainly seen in Africa, the Middle East, and Asia. In the U.S., about 10% of blacks have G6PD deficiency. Exacerbating factors - G6PD deficiency is usually asymptomatic unless the affected individual consumes a drug or food (fava beans) that increases oxidative stress. Common drugs that can cause hemolysis in G6PD deficiency include sulfonamides, primaquine, dapsone, and nitrofurantoin. A website that contains a list of drugs associated with G6PD-mediated hemolysis is available here - G6PD drug list. G6PD deficiency can also cause prolonged jaundice in neonates. [46] Hereditary spherocytosis Pathology - hereditary spherocytosis is caused by inherited defects in the RBC membrane. The defects allow sodium and water to enter the cell which causes it to assume the shape of a sphere. Spherocytes are hemolyzed in the spleen by macrophages. The clinical sequelae of the disease is broad and depends upon the defect that is inherited. Mild disease (20 - 30% of cases) is often asymptomatic and severe disease (3 - 5% of cases) may cause life-threatening anemia. Most patients have moderate disease (60 - 70%) which presents with moderate anemia, splenomegaly, and jaundice in childhood. Prevalence - the prevalence of hereditary spherocytosis in the U.S is about 0.02 - 0.05% Treatment - splenectomy is curative in almost all cases. [47] Sickle cell anemia The abnormal RBCs that occur in sickle cell anemia are hemolyzed in the spleen by macrophages. Intravascular hemolysis also occurs. See sickle cell anemia for more. Thalassemias The abnormal hemoglobin that occurs in the thalassemias can lead to RBC deformities and hemolysis. See alpha thalassemia and beta thalassemia above.

• Diagnosis
• Hemolytic anemia should be considered in any patient with risk factors for the condition
• The table below details the labs that are used to evaluate for hemolytic anemia, and their findings depending on the cause

• References [39,40,42,43,44,45,46,47]

Labs to detect hemolysis
Reticulocyte count - the reticulocyte count should be increased in hemolysis unless there is an underlying condition that is suppressing hematopoiesis LDH - The LDH level is one of the more useful tests for diagnosing hemolysis. Elevated LDH in combination with a haptoglobin level < 25 mg/dL is 95% specific for the presence of hemolysis while a normal LDH and haptoglobin > 25 mg/dL are 92% sensitive for the absence of hemolysis. Haptoglobin - The haptoglobin level is one of the more useful tests for diagnosing hemolysis. Elevated LDH in combination with a haptoglobin level < 25 mg/dL is 95% specific for hemolysis while a normal LDH and haptoglobin > 25 mg/dL are 92% sensitive for the absence of hemolysis. Unconjugated bilirubin - unconjugated (indirect) bilirubin levels may be elevated in hemolysis, but are rarely > 3 mg/dl unless significant liver disease is present Urinalysis - bilirubin, urobilinogen, and hemosiderin may be present but are typically only seen in severe intravascular hemolysis
Autoimmune hemolytic anemia labs
Direct antiglobulin test (DAT, direct "Coombs test") - the direct antiglobulin test detects antibodies (IgG) and complement (C3) attached to RBCs. IgG attached to RBCs is seen in WAHA. C3 attached to RBCs can occur in WAHA, but it is more common in CAHA. The DAT test is not specific and may be positive in people without AHA (up to 8% of hospitalized patients) and negative in up to 10% of patients with AHA. Peripheral smear - in AHA, the peripheral smear will typically show signs of reticulocytosis (polychromasia and anisocytosis). Spherocytes (formed when complement fixation increases cell permeability) are seen in WAHA and RBC agglutination is seen in CAHA. Cold agglutinin titer - if CAHA is suspected, a cold agglutinin titer may be useful. Cold agglutinins are IgM antibodies that can cause RBC agglutination, and they are most reactive at 39.2° F.
Mechanical hemolysis labs
Peripheral smear - in mechanical hemolysis, schistocytes (RBC fragments) are often seen on the peripheral smear ADAMTS13 activity - low activity makes TTP more likely; can help distinguish TTP from HUS and other similar conditions ADAMTS13 antibody - detects IgG antibodies to ADAMTS13; helps to diagnose TTP and distinguish acquired TTP (positive antibodies) from inherited TTP (no antibodies) Urinalysis - proteinuria, RBCs and RBC casts may be seen in HUS Stool culture - stool culture for E. coli 0157:H7 and Shigella bacteria when HUS is suspected Complement levels - low complement levels may help in the diagnosis of HUS Clotting times (aPTT and PT) - prolonged clotting times will be seen in DIC. Clotting times may be slightly increased in TTP. D-dimer - D-dimer levels will be elevated in DIC. They may be slightly increased in TTP. Fibrinogen - may be decreased in DIC, but is is also an acute phase reactant and up to 57% of DIC patients having normal levels Platelet count - platelet counts will be low in TTP and DIC
RBC abnormality labs
Peripheral smear - Heinz bodies and bite cells are seen in G6PD deficiency along with hemighosts (RBCs that have unevenly distributed hemoglobin). Spherocytes are seen in hereditary spherocytosis. Sickle cells are seen in sickle cell anemia. G6PD activity - G6PD reduces NADP to NADPH and measurement of this reaction is used to quantify G6PD activity in RBCs Mean corpuscular hemoglobin concentration (MCHC) - because of their irregular shape, hemoglobin in spherocytes is concentrated, so the MCHC will be elevated in hereditary spherocytosis. Mean corpuscular volume (MCV) - RBCs are small in the thalassemias so the MCV is reduced

Autoimmune hemolytic anemia treatment
Overview Treatment depends upon the underlying etiology and severity of anemia. In cases where infections or drugs are the inciting factors, treatment or resolution of the infection and removal of the offending drug often leads to remission. When the underlying condition is not transient (e.g. cancer, SLE, immunodeficiencies), treatment of the condition may improve AHA, but it does not always lead to remission. Primary WAHA is typically severe and spontaneous remission is uncommon. Primary CAHA or "cold agglutinin disease" typically causes mild to moderate anemia that can be exacerbated by cold temperatures, infections, and surgery. WAHA and CAHA increase the risk of thrombosis with up to 11% of affected patients experiencing a thrombotic event in studies Patients with mild anemia can be monitored while patients with significant anemia will require treatment Folic acid supplementation of 1 - 5 mg/day is recommended because active hemolysis can consume folate and lead to macrocytosis WAHA treatment Corticosteroids (first-line) - typically prednisone 1 mg/kg/day for 2 - 3 weeks then taper with goal to stop within 3 - 6 months; median response time is 7 days; 80% of patients will respond; sustained remission occurs in about 30% of patients Rituximab (second-line) - 375 mg/m2 weekly for 4 weeks; may be combined with corticosteroids; median response time is 3 - 6 weeks; around 79% of patients will respond; sustained remission has been seen in up to 70% of patients Splenectomy (third-line) - 70% response rate with 40% achieving complete remission VTE prophylaxis - all hospitalized patients should receive VTE prophylaxis; consider VTE prophylaxis in ambulatory patients at increased risk CAHA treatment Asymptomatic - patients with Hg ≥ 10 g/dl can be monitored; folic acid 1 - 5 mg/day should be given if chronic hemolysis is present Symptomatic - consider rituximab, plasmapheresis, eculizumab, and RBC transfusions. An antibody that targets C1 and prevents complement fixation was FDA-approved in 2022 to treat cold agglutinin disease. It is called sutimlimab (Enjaymo®), and in a trial involving 24 patients, it was shown to be effective in preventing cold agglutinin-induced hemolysis. [PMID 33826820] [Enjaymo® PI] VTE prophylaxis - all hospitalized patients should receive VTE prophylaxis; consider VTE prophylaxis in ambulatory patients at increased risk [38,40,58]
Mechanical hemolysis treatment
Artificial heart valves - beta blockers may reduce shear forces and improve hemolysis. Pentoxifylline decreases blood viscosity and may improve hemolysis. In severe cases, paravalvular leak repair may be necessary. [42] Thrombotic thrombocytopenic purpura (TTP) - the primary treatment is plasma exchange which replaces the deficient ADAMTS13 enzyme. A drug called caplacizumab (Cablivi®) was FDA-approved in 2019 to treat TTP. Caplacizumab is an antibody that inhibits the interaction between von Willebrand factor and platelets. [Cablivi® PI] Immunosuppressants such as corticosteroids are also frequently given. [43] Hemolytic uremic syndrome (HUS) - treatment of Shiga-like toxin-associated HUS is supportive. Atypical HUS can be treated with drugs that inhibit the complement system (eculizumab and ravulizumab) and plasma exchange. [43,44] Disseminated intravascular coagulation (DIC) - the main treatment for DIC is to correct the underlying disorder. Significant bleeding is treated with replacement of platelets and clotting factors (fresh frozen plasma). If bleeding is not significant, heparin may be used to prevent thrombosis. [45]
RBC abnormality treatment
Paroxysmal nocturnal hemoglobinuria (PNH) - PNH is treated with eculizumab (Soliris), an anti-complement antibody [41] Glucose-6-phosphate dehydrogenase (G6PD) deficiency - Treatment of G6PD deficiency is supportive, and it typically resolves once the inciting factor is removed [46] Hereditary spherocytosis - splenectomy [47] Sickle cell anemia - see sickle cell treatment above Thalassemia - see alpha thalassemia and beta thalassemia above

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• MACROCYTOSIS

• Overview
• Macrocytosis, defined as MCV > 100 fL, is present in about 3% of the population. Macrocytosis can be divided into two subtypes: megaloblastic and nonmegaloblastic.


• Megaloblastic macrocytosis
• Megaloblastic macrocytosis occurs when there is insufficient folic acid and/or vitamin B12 during erythropoiesis. Folic acid and vitamin B12 are essential for the production of DNA, and their deficiency causes RBCs to develop abnormally and become enlarged. Hypersegmented neutrophils (neutrophils with six or more lobes or the presence of more than 3% of neutrophils with at least five lobes) are often seen in megaloblastic macrocytosis and their presence can be helpful in determining the subtype.


• Nonmegaloblastic macrocytosis
• The mechanisms behind nonmegaloblastic macrocytosis are varied and not completely understood. Common causes of nonmegaloblastic macrocytosis are alcohol abuse, medications, and hypothyroidism.

• References [48, 49, 53]

Megaloblastic causes
Vitamin B12 deficiency See vitamin B12 deficiency below
Folate/folic acid deficiency (Vitamin B9) Folate is a naturally-occurring vitamin found in dark leafy green vegetables, citrus fruits, and liver. Folic acid is a synthetic version of folate that is found in supplements and fortified foods such as breads and cereals. Folic acid is essential for the production of DNA. Folic acid deficiency causes RBCs to develop abnormally leading to enlarged RBCs called macrocytes. Macrocytes carry oxygen normally, but they are not as pliable as normal RBCs and their lifespan is decreased by 30 - 50%. Folate deficiency is typically functional or related to decreased absorption. Dietary deficiency is rare with the exception of alcoholics. Medications can affect folate metabolism and absorption. See drugs that affect DNA/RNA metabolism and drugs that decrease folic acid absorption for more. Intestinal diseases such as celiac and inflammatory bowel disease can cause decreased absorption, and increased consumption can occur in pregnancy, lactation, and hemolytic anemia. Serum folic acid levels fluctuate widely with daily dietary intake, and they are not a good measure of folate reserves. RBC folate is a test that measures the amount of folate in RBCs and is a better reflection of folate tissue stores. RBC folate will begin to decline after about 4 months of deficient folate intake.
Nonmegaloblastic causes
Alcohol abuse Most common cause (up to 80%) in many populations Mechanism behind macrocytosis is not completely understood but is thought to occur through the direct toxic effect of alcohol on erythropoiesis. Cessation of drinking corrects macrocytosis. Significant alcoholism may also have associated folic acid deficiency
Hemolysis / Hemorrhage Hemolysis and hemorrhage can lead to reticulocytosis. Reticulocytes are 20% larger than mature RBCs and when their percentage is increased, the average MCV also increases.
Hypothyroidism Common cause in older individuals
Medication side effects A number of medications have been associated with macrocytosis. See drugs associated with macrocytosis below
Myelodysplasia Myelodysplasia is a more common cause among elderly patients. The peripheral smear may show signs of myelodysplasia but a bone marrow biopsy is required for diagnosis.
Liver disease Liver disease can lead to the deposition of cholesterol or phospholipids on the surface of RBCs which increases their surface area and makes them enlarged
COPD Macrocytosis in COPD is thought to occur because of excessive cell water that is secondary to carbon dioxide retention
Splenectomy The spleen normally removes lipids from the maturing RBC membrane. After splenectomy, this removal is decreased and the RBC membrane surface area is increased causing enlargement.
Spurious causes
Cold agglutinins Cold agglutinins can cause RBCs to clump making them appear larger to the hemolyzer than they really are
Hyperglycemia When hyperglycemic blood is diluted, RBCs may swell more than usual causing a false elevation in MCV
Significant leukocytosis Increased turbidity may cause the machine to overestimate MCV

• References [49, 53]

Megaloblastic vs Nonmegaloblastic
Peripheral smear Hypersegmented neutrophils (neutrophils with six or more lobes or the presence of ≥ 3% of neutrophils with at least five lobes) are often present in megaloblastic macrocytosis. They may also be seen in myelodysplasia. Oval-shaped macrocytes are suggestive of megaloblastic macrocytosis and round macrocytes suggest liver or bone marrow disease Target cells may be present in liver disease or after splenectomy An increase in reticulocytes can indicate a nonmegaloblastic macrocytosis such as hemolytic anemia Reticulocyte count An elevated reticulocyte count (> 4%) can indicate a nonmegaloblastic macrocytosis secondary to hemolytic anemia. See hemolytic anemia above. Vitamin B12 level Levels > 400 pg/ml are consistent with sufficient vitamin B12 for erythropoiesis Levels 100 - 400 pg/ml are borderline low and may require further evaluation with methylmalonic acid and homocysteine See vitamin B12 diagnosis for more Methylmalonic acid (MMA) levels Methylmalonic acid (MMA) is a byproduct of protein metabolism that is converted to succinyl-coenzyme A. Vitamin B12 is a cofactor in MMA conversion, and if vitamin B12 levels are low, conversion is decreased and MMA levels rise. Elevated MMA levels are an early predictor of vitamin B12 deficiency. Elevated MMA levels with elevated homocysteine is indicative of vitamin B12 deficiency whereas normal MMA levels and elevated homocysteine levels is suggestive of folic acid deficiency Homocysteine levels Homocysteine is an intermediary amino acid that is converted to methionine through a reaction that requires folic acid and vitamin B12. Homocysteine levels will rise in the setting of deficient folic acid and/or vitamin B12. Elevated homocysteine levels are an early and sensitive indicator of folic acid and vitamin B12 deficiency. Elevated MMA levels with elevated homocysteine is indicative of vitamin B12 deficiency whereas normal MMA levels and elevated homocysteine levels is suggestive of folic acid deficiency Folic acid/RBC folate levels Serum folate levels fluctuate widely with daily dietary intake, and they are not a good measure of folate reserves RBC folate is a test that measures the amount of folate in RBCs and is a better reflection of folate tissue stores. RBC folate will begin to decline after about 4 months of deficient folate intake.
Nonmegaloblastic labs
Bone marrow biopsy - to look for myelodysplasia TSH - to rule out hypothyroidism Liver function tests - to look for liver disease LDH and haptoglobin - if hemolytic anemia is suspected Peripheral smear - target cells in liver disease; signs of hemolysis Reticulocyte count - elevated in hemolysis

• Reference [48,49]

Drugs associated with macrocytosis
Drugs that interfere with DNA/RNA metabolism and/or synthesis
Allopurinol Azathioprine (Imuran®) Capecitabine Cladribine (Mavenclad®) Cyclophosphamide Cytosine arabinoside Fludarabine Fluorouracil Gadolinium (MRI contrast) Gemcitabine HIV reverse transcriptase inhibitors (e.g. stavudine, lamivudine, zidovudine) Hydroxyurea Leflunomide (Arava®) Mercaptopurine Methotrexate Mycophenolate Nitrous oxide Pentostatin Pyrimethamine Teriflunomide (Aubagio®) Thioguanine Trimethoprim (Bactrim®) Tyrosine kinase inhibitors (eg, sunitinib, and imatinib) Valproic acid (Depakote®)
Drugs that decrease folic acid absorption
Alcohol Aminopterin Aminosalicylates (Sulfasalazine, Mesalamine, etc.) Artemether lumefantrine Birth control pills Chloramphenicol Chloroquine Cholestyramine (Questran®) Estrogens Erythromycin H2 blockers (e.g. Pepcid®) Metformin Nitrofurantoin (Macrobid®) Penicillins Phenobarbital Phenytoin Primaquine Proton pump inhibitors (e.g. omeprazole, Nexium®) Quinine Sulfadoxine–pyrimethamine Tetracyclines
Drugs that decrease vitamin B12 absorption
Aminosalicylates (Sulfasalazine, Mesalamine, etc.) Colchicine (Colcrys®, Mitigare®) Cycloserine H2 blockers (e.g. Pepcid®) Isoniazid Metformin Proton pump inhibitors (e.g. omeprazole, Nexium®)

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• VITAMIN B12 DEFICIENCY

• Physiology
• Vitamin B12 is an essential cofactor in the synthesis of DNA. Rapidly dividing cells like RBCs require adequate amounts of vitamin B12 to carry out erythropoiesis. Insufficient vitamin B12 levels cause RBCs to develop abnormally resulting in enlarged cells (macrocytes) with flimsy membranes. Macrocytes carry oxygen normally, but their fragility shortens their lifespan by one-half to one-third.
• Daily requirements of vitamin B12 to support normal erythropoiesis are about 1 - 3 mcg. The liver stores about 1000 times this amount so it takes about 3 - 4 years of deficient absorption before macrocytosis and anemia develop.
• Vitamin B12 absorption occurs through a series of steps. First, parietal cells in the gastric mucosa secrete a glycoprotein called intrinsic factor that binds vitamin B12 in food and protects it from digestion. The B12-intrinsic factor complex then travels to the terminal ileum where it is absorbed and transported to the liver for storage.

• Causes
• Atrophic gastritis (pernicious anemia) - atrophic gastritis results from autoimmune antibodies that destroy gastric parietal cells and lead to the loss of intrinsic factor. Pernicious anemia typically affects older patients with a median age of diagnosis between 70 - 80 years.
• Medications - see drugs that decrease B12 absorption for more
• Bowel surgery - bowel surgeries that affect the stomach (e.g. gastric bypass) and terminal ileum can lead to decreased B12 absorption
• Intestinal diseases - diseases that affect the lining of intestine (e.g. celiac disease, Crohn's, helminth infections) can reduce the absorption of vitamin B12
• Vegetarian diet - vitamin B12 is mainly found in animal products (fish, meat, chicken, eggs, dairy). Strict vegetarians who do not take supplements are at risk for deficiency.
• Chronic pancreatitis - pancreatic enzymes are necessary for the proper processing of vitamin B12 in the duodenum and their deficiency can lead to decreased absorption
• H pylori infection - in rare cases, H pylori infection may cause gastritis that leads to decreased intrinsic factor

• Symptoms
• Because vitamin B12 is necessary for DNA synthesis, vitamin B12 deficiency affects rapidly dividing cells like RBCs, skin cells, and cells of the mucous membranes. Vitamin B12 is also essential for the maintenance of the myelin sheaths that surround nerve cells. Symptoms of vitamin B12 deficiency include anemia, paresthesias, inflammation of the lips and gums, difficulty walking, and loss of position and vibration sense.

• Diagnosis
• Vitamin B12 level
• Levels > 400 pg/ml are consistent with sufficient vitamin B12 for erythropoiesis
• Levels 100 - 400 pg/ml are borderline low and may require further evaluation with methylmalonic acid and homocysteine


• Methylmalonic acid (MMA) levels - Methylmalonic acid (MMA) is a byproduct of protein metabolism that is converted to succinyl-coenzyme A. Vitamin B12 is a cofactor in MMA conversion, and if vitamin B12 levels are low, conversion is decreased and MMA levels rise. Elevated MMA levels are an early predictor of vitamin B12 deficiency. Elevated MMA levels with elevated homocysteine is indicative of vitamin B12 deficiency whereas normal MMA levels and elevated homocysteine levels is suggestive of folic acid deficiency.


• Homocysteine levels - Homocysteine is an intermediary amino acid that is converted to methionine through a reaction that requires folic acid and vitamin B12. Homocysteine levels will rise in the setting of deficient folic acid and/or vitamin B12. Elevated MMA levels with elevated homocysteine is indicative of vitamin B12 deficiency whereas normal MMA levels and elevated homocysteine levels is suggestive of folic acid deficiency.


• Peripheral smear - the presence of hypersegmented neutrophils (neutrophils with six or more lobes or the presence of ≥ 3% of neutrophils with at least five lobes) is suggestive of megaloblastic macrocytosis. Macroovalocytes are also suggestive of megaloblastic macrocytosis.

• Intrinsic factor antibodies - intrinsic factor antibodies are seen in pernicious anemia. There are 2 types of antibodies. Type 1 antibodies block the binding or vitamin B12 to intrinsic factor. Type 2 antibodies block ileal intrinsic factor binding sites. Type 1 antibodies are present in 50% of patients with pernicious anemia and Type 2 antibodies are seen in 35%. Intrinsic factor antibodies have a sensitivity of 50% and specificity of 100% for pernicious anemia.

• Gastric parietal cell antibodies - parietal cell antibodies are present in 90% of patients with pernicious anemia. They are also found in up to 60% of patients with chronic atrophic gastritis who do not have vitamin B12 malabsorption, 30% of patients with Sjogren's syndrome, 20% of patients with gastric ulcers, and 7% of healthy adults. Parietal antibodies have a sensitivity of 90% for pernicious anemia but their specificity varies widely depending on the population tested.

• Treatment
• The treatment for vitamin B12 deficiency is replacement. Vitamin B12 is available in oral forms, a prescription nasal spray, and intramuscular injections. If absorption is still intact or only mildly affected, then oral supplementation is usually sufficient. If absorption is significantly impaired, then the intramuscular route is preferred. Trials have shown that even in patients with pernicious anemia or ileal resection, oral supplementation is often effective.
• Megaloblastic anemia typically responds after 6 - 8 weeks of treatment. Resolution of neurologic symptoms depends upon the severity and can take weeks to months.
• Vitamin B12 for injection is called cyanocobalamin in the U.S. and hydroxocobalamin in Europe. Oral forms of vitamin B12 (cyanocobalamin and methylcobalamin) are available from a number of supplement manufacturers. An intranasal product called Nascobal® is also available by prescription but is very expensive and rarely prescribed. Common treatment regimens are presented below.
• Cyanocobalamin injection - administer 1000 mcg intramuscularly once daily or every other day for 1 week, then once weekly for 4 - 8 weeks, then once monthly from then on
• Oral vitamin B12 (cyanocobalamin) 500 - 2000 mcg once daily
• Dosing to prevent deficiency in vegans - supplement containing ≥ 2 mcg of vitamin B12 once daily [49, 53, 54, 55]

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• ANEMIA IN THE ELDERLY

• Overview
• Anemia is more common in older populations with a prevalence of 10% in community dwelling adults ≥ 65 years old. In patients ≥ 85 years old, the prevalence jumps to more than 20%. Anemia also varies by race. In a cross-sectional study of persons ≥ 65 years old, the prevalence of anemia was as follows: Blacks - 28%, Mexican-Americans - 10.4%, Whites - 9%, Other races - 14%. The racial disparity probably reflects the higher prevalence of anemia-causing conditions (e.g. thalassemia) that affect certain races more than others.
• The etiology of anemia in older patients is diverse, and in many cases, a cause is never found. The lack of a definitive diagnosis in a significant number of patients led to the creation of a entity called "unexplained anemia of the elderly" which is defined as a normocytic anemia that is not secondary to nutritional deficiency, kidney disease, or inflammatory disease and is characterized by a blunted response to erythropoietin. [50,52]
• The results below are from a study where 174 anemic elderly adults (median age 76 years) underwent an extensive workup to determine a cause

• ^✝Other causes included the following: hemolytic anemia (4 cases), alcohol (3 cases), hypothyroidism (1 case), drug-induced (1 case), vitamin B12 deficiency (1 case),
• Reference [51]

Cause of anemia in 174 elderly patients
Cause	% of patients
Unexplained anemia of the elderly	44%
Iron-deficiency anemia	25%
Anemia of inflammation	9.8%
Hematologic malignancy	7.5%
Other causes✝	5.7%
Thalassemia	4.6%
Chronic kidney disease	3.4%

• Diagnosis
• The workup for anemia in elderly patients is the same as younger patients with the understanding that a significant number of patients (up to 44%) will have no identifiable cause
• Iron-deficiency anemia is the most common identifiable cause, and in some studies, up to 22% of patients without laboratory findings consistent with iron-deficiency anemia responded to oral iron therapy. If iron deficiency is present, a gastrointestinal workup should be performed, keeping in mind that up to 66% of these patients will have a negative workup. IV iron may be necessary in patients where decreased iron absorption is thought to be the underlying cause.
• If the GFR is < 60 ml/min, chronic kidney disease should be considered, but it is not a common cause until the GFR falls below 30 ml/min
• When an extensive workup yields no identifiable cause, undetermined anemia of the elderly should be considered [52]

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• IRON REPLACEMENT

• Adult oral iron replacement
• Adults with iron deficiency are typically treated with 65 - 195 mg of elemental iron a day (Ferrous sulfate 325 mg one to three times a day)
• Several recent studies have found that less frequent iron dosing leads to greater absorption. In one study, lower iron doses (40 - 80 mg elemental iron) given once daily as opposed to twice daily resulted in better absorption. [PMID 26289639]. Another study found that iron absorption was significantly greater with every other day dosing when compared to daily dosing. [PMID 29032957]
• It is sometimes recommended that a vitamin C supplement be taken with iron to enhance its absorption. A study published in 2020 found that taking vitamin C with iron did not improve hemoglobin or ferritin levels when compared to iron alone. [PMID 33136134]
• Hemoglobin levels should rise by about 2 g/dL every 3 weeks
• Once hemoglobin levels are normal, iron replacement should continue for 3 - 6 months to replenish iron stores [5,9]

• Pediatric oral iron replacement
• Treat iron deficiency with iron drops at a dose of 3 mg/kg/day of elemental iron. Dose is given in 1 - 2 divided doses a day.
• Repeat hemoglobin testing in 4 weeks. An increase in hemoglobin concentration of ≥ 1 g/dL or in hematocrit of ≥ 3% confirms the diagnosis of iron-deficiency anemia.
• If iron-deficiency anemia is confirmed, reinforce dietary counseling, continue iron treatment for 2 more months, then recheck hemoglobin concentration or hematocrit
• Reassess hemoglobin concentration or hematocrit approximately 6 months after successful treatment is completed [4]
• A study published in 2017 found that ferrous sulfate was superior to iron polysaccharide complex in treating nutritional iron-deficiency anemia in children aged 9 - 48 months. [PMID 28609534]

• Oral iron products
• There are three basic forms of iron that are available in supplements
• Each form of iron contains a different percentage of elemental iron
• Modified release products are not recommended because they likely decrease iron absorption

• Side Effects
• Common side effects of iron supplements are listed below
• Taking iron after a meal may lessen side effects. Gradually increasing the dose may also help to minimize side effects.
• Iron supplements do NOT affect guaiac (Hemoccult II® and Sensa®) stool testing [3, 6]
• Common side effects of iron supplements include:
• Constipation
• Nausea
• Stomach upset
• diarrhea
• Dark-colored stools

• IV iron replacement
• IV iron replacement may be necessary in some patients, particularly when impaired absorption is an issue or when rapid correction is desired. IV iron also enhances the effects of erythropoietin agents.
• Hypersensitivity infusion reactions have traditionally limited the use of IV iron, but newer formulations are better tolerated and have led to expanded use, although the high cost of IV iron is still an issue
• The amount of iron that can be infused during a single treatment depends on the product and ranges from 250 - 1000 mg
• Side effects of IV iron include itching, nausea and vomiting, muscle cramping, chest pain, headache, and flushing. Serious hypersensitivity reactions are rare. [9]
• Dosing for IV iron is calculated with the following formula:
• Body weight(kg) X 2.3 X hemoglobin deficiency + 500 to 1000 mg iron (for the repletion of iron stores)
• Where hemoglobin deficiency = target hemoglobin level - patient hemoglobin level

• Indications for IV iron
• Failure of oral iron therapy
• Impaired absorption of oral iron (see risk factors for iron deficiency above)
• Need for rapid correction (e.g. pregnancy)
• Individual objection to blood transfusion (e.g. Jehovah's Witness)
• Use of erythropoietin agents in chronic kidney disease [9]

• Recommended dietary iron intake
• An adult male contains about 50 mg/kg of iron. An adult menstruating female has about 40 mg/kg of iron.
• Median dietary intake of iron is about 16 - 18 mg/day in men and 12 mg/day in women. Healthy adults absorb about 10 - 15% of dietary iron
• Adult men need to absorb about 1 mg/day of iron to maintain iron balance. Adult menstruating females need to absorb about 1.5 mg/day to maintain iron balance. There is high individual variation in daily iron requirements. In the absence of bleeding, only a small amount of iron is lost from the body each day (0.90 - 1.02 mg/day).
• In food sources, iron is found in two forms - heme iron and nonheme iron
• Nonheme iron is the primary source of iron absorbed from food. A small amount of heme iron is absorbed. [1]

• * Human breast milk has more bioavailable iron than formula. Iron in cow's milk is poorly absorbed.
• In the U.S., cow's milk is not recommended until the age of one year
• Reference [1]

U.S. recommended dietary iron intake
Age	Male (mg/day)	Female (mg/day)
0 - 6 months	0.27*	0.27*
7 - 12 months	11	11
1 - 3 years	7	7
4 - 8 years	10	10
9 - 13 years	8	8
14 - 18 years	11	15
19 - 30 years	8	18
31 - 50 years	8	18
≥ 51 years	8	8
Pregnancy	-	27

• Heme iron
• Heme iron is found in animal products that contain hemoglobin such as red meat, fish, and poultry products. It is absorbed more easily than nonheme iron. About 15 - 35% of heme iron is absorbed from food. Heme iron is not typically affected by other dietary components.
• The iron content of a large number of foods can be found here - USDA food database

• Reference [6]

Food sources of heme iron
Food	Iron content per 1 serving (3 ounces)
Chicken liver	11 mg
Canned oysters	5.7 mg
Beef liver	5.2 mg
Beef chuck	3.1 mg
Turkey, dark meat	2.0 mg
Beef, ground	2.2 mg
Beef, top sirloin	1.6 mg
Tuna, light	1.3 mg
Turkey, light meat	1.1 mg
Chicken, dark meat	1.1 mg
Chicken, light meat	0.9 mg
Pork, loin chop	0.7 mg
Halibut	0.2 mg

• Nonheme iron
• Nonheme iron is found in fruits and vegetables. It is also in many grain and cereal products which are iron-fortified. About 2 - 20% of nonheme iron is absorbed. Nonheme iron absorption is affected by other dietary components. Nonheme iron is the most abundant source of iron in most diets. [1,6]
• The iron content of a large number of foods can be found here - USDA food database

• Reference [6]

Food sources of nonheme iron
Food (serving size)	Iron content per 1 serving
Iron-fortified cereal (3/4 cup)	18 mg
Oatmeal, instant (1 packet)	11 mg
Soybeans (1 cup)	8.8 mg
Beans, kidney (1 cup)	5.2 mg
Beans, lima (1 cup)	4.5 mg
Black eyed peas (1 cup)	4.3 mg
Beans, navy (1 cup)	4.3 mg
Beans, black (1 cup)	3.6 mg
Beans, pinto (1 cup)	3.6 mg
Tofu (1/2 cup)	3.4 mg
Spinach, fresh (1/2 cup)	3.2 mg
Raisins (1/2 cup)	1.6 mg
Bread, white (1 slice)	0.9 mg
Bread, whole-wheat (1 slice)	0.7 mg

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• IRON LABS

• Ferritin
• Ferritin is the major storage form of iron in the body (see RBC and iron homeostasis for more). The liver, spleen, and bone marrow are the primary sites of iron storage.
• Ferritin is mostly found in cells while a small amount circulates in the blood. Ferritin concentrations in the blood are proportional to cellular concentrations which makes serum ferritin levels a good measure of body iron stores. Serum ferritin is the most sensitive and specific test for detecting iron deficiency.
• In adults, 1 ng/ml of serum ferritin represents about 8 mg of storage iron. Total body iron stores normally range from 600 mg - 1500 mg in non-menstruating adults.
• Ferritin levels < 30 ng/ml are considered iron deficient regardless of hemoglobin status. When serum ferritin concentrations fall below 12 ng/ml, iron stores are totally depleted.
• Ferritin is also an "acute phase reactant" which means inflammatory states (ex. infections, cancer, liver disease) may cause an increase in serum concentrations. In anemia of inflammation, ferritin levels may be normal when an iron-deficient state is actually present. [1,9]
• For adults living in the U.S., the median serum ferritin concentrations are 36 - 40 ng/ml for menstruating females, and 112 - 156 ng/ml for men [1]
• Normal ranges
• Adult male: 30 - 400 ng/ml
• Adult female: 15 - 150 ng/ml
• Reference: LabCorp®
• Factors which may cause a spuriously high ferritin level (normal ferritin when iron stores are low)
• Infections
• Cancer
• Liver disease
• Increased alcohol consumption
• Obesity
• Diabetes

• Serum iron
• The serum iron level is the amount of iron that is present in the blood
• Iron levels are used with the TIBC to calculate the transferrin saturation which is a more useful indicator of iron status
• Ferritin is a more useful measure of total body iron stores.
• Normal ranges:
• Adult male: 45 - 170 μg/dL
• Adult female: 40 - 160 μg/dL
• Reference: Quest Diagnostics®
• Abnormal values:
• Low serum iron
• Low iron levels are typically seen in iron deficiency. They may also be seen in chronic disease states.
• High serum iron
• Hemochromatosis
• Hemolytic anemia
• Sideroblastic anemia
• Iron poisoning

• Soluble transferrin receptor
• Transferrin receptor is a transmembrane protein that regulates iron uptake by cells. Cellular expression of transferrin receptor is increased when a cell needs iron. Soluble transferrin receptor is a cleavage product of transferrin receptor that can be measured in the blood.
• Erythroid progenitor cells are the primary cells that need iron, and 80% of soluble transferrin receptor in the blood comes from these cells. Inflammation does not affect soluble transferrin receptor levels, so this makes it a specific marker for iron deficiency. This lab is not widely available.
• Anemia of inflammation
• Levels are typically normal
• Iron-deficiency anemia
• Levels are increased

• Total Iron Binding Capacity (TIBC)
• When iron is transported in the blood between cells, it is mostly bound to transferrin (see RBC and iron homeostasis for more)
• Transferrin can be measured directly, but it is commonly measured indirectly with the Total Iron Binding Capacity. To measure TIBC, iron is added to a sample of blood so that all the transferrin is saturated with iron. The TIBC is then calculated.
• The TIBC test is used in conjunction with the total serum iron level to calculate the transferrin saturation or "% saturation" which represents the amount of transferrin that is saturated with iron
• Normal ranges
• Adult male: 250 - 425 μg/dL
• Adult female: 250 - 450 μg/dL
• Reference: Quest Diagnostics®
• Abnormal values
• High TIBC
• A high TIBC usually indicates iron deficiency. It can also be increased in pregnancy and with oral contraceptive use.
• Low TIBC
• Hemochromatosis
• Malnutrition
• Chronic disease
• Inflammation
• Liver disease
• Nephrotic syndrome [2]

• Transferrin saturation (TSAT)
• The transferrin saturation is the proportion of transferrin (expressed as a percentage) that is saturated with iron
• Transferrin saturation below 16% indicate that the rate of delivery of iron is insufficient to maintain the normal rate of hemoglobin synthesis
• For adults living in the U.S., the median transferrin saturations are 26 - 30% for men, and 21 - 24% for women [1]
• Transferrin saturation is calculated with the following formula:
• Transferrin saturation (%) = (Serum iron level / TIBC) X 100
• Normal ranges
• Adult male: 20 - 50%
• Adult female: 15 - 50%
• Reference: Quest Diagnostics®
• Abnormal values
• Low saturation
• A low transferrin saturation is typically seen in iron deficiency. It may also be seen in chronic disease states.
• High saturation
• Hemochromatosis
• Hemolytic anemia
• Sideroblastic anemia
• Iron poisoning

• Unbound Iron Binding Capacity (UIBC)
• The UIBC is the amount of transferrin that is not bound by iron
• It is measured with the following formula:
• UIBC(μg/dL) = TIBC(μg/dL) - Serum Iron(μg/dL)

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• BLOOD DONATION

• Overview
• People who donate blood frequently may be at increased risk of iron deficiency
• A study published in 2015 looked at the effects of iron supplementation after blood donation on hemoglobin recovery. The study is detailed below.

• STUDY
  Oral iron supplementation after blood donation: a randomized clinical trial, JAMA (2015) [PubMed abstract]
• A study published in the JAMA in 2015 looked at the effects of iron supplementation after blood donation
• The study enrolled 215 patients who had not donated blood within the previous 4 months
• Patients were stratified by their baseline ferritin levels: low ferritin ≤ 26 ng/ml | higher ferritin > 26 ng/ml
• Every patient donated a unit of blood (500 ml) after which they were randomized to ferrous gluconate 325 mg (37.5 mg elemental iron) once daily or no iron (control)
• The primary outcome was hemoglobin recovery, defined as the time for restoration of 80% of the hemoglobin lost at donation
• During follow-up, the following was seen:
• After donation hemoglobin decline: low ferritin - 13.4 to 12 g/dl | higher ferritin - 14.2 to 12.9 g/dl
• Average time to primary outcome in the iron group: low ferritin - 32 days | higher ferritin - 31 days
• Average time to primary outcome in the control group: low ferritin - 158 days | higher ferritin - 78 days
• Median time to recover to baseline ferritin level in the iron group: low ferritin - 21 days | higher-ferritin - 107 days
• Median time to recover to baseline ferritin level in the control group: low ferritin >168 days | higher ferritin >168 days

• Summary
• In the U.S., individuals are allowed to donate blood every 8 weeks
• This study showed that on average, most people do not recover iron stores and hemoglobin levels in that time period
• Iron supplementation with 325 mg of ferrous gluconate once daily had a profound effect on hemoglobin and iron store recovery
• Patients at increased risk of anemia and frequent blood donors should consider taking an iron supplement for 2 - 4 months post-donation

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