ANEMIA AND HEMOGLOBINOPATHIES













Red blood cell life cycle and iron homeostasis



  • 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, then iron deficiency is the likely predominant cause.
    • Ferritin > 100 mcg/L and/or TSAT > 20%
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, then iron deficiency is the likely predominant cause.
      • If GFR is < 60 ml/min and reticulocyte count is inappropriate for the degree of anemia, consider anemia of chronic kidney disease
      • If reticulocyte count is elevated, consider hemolytic anemia
MCV is elevated (> 100 fL)







Hemoglobin illustration
  • A total of 4 genes (2 on each chromosome 16) code for alpha globulin. A total of 2 genes (1 on each chromosome 11) code for beta globulin.
  • References [12, 15 - 21]
HEMOGLOBIN TYPE BY CONDITION
Condition Hg A Hg A2 Hg F Hg S/C/H/Bart Clinical Prevalence
Normal 95 - 98% 1.5 - 3.0% 0.5% 0 - -
Alpha (α) Thalassemias
Condition Hg A Hg A2 Hg F Hg H Hg Bart Clinical Affected populations
α-thalassemia carrier
(1/4 genes affected)
95 - 98% 1.5 - 3.0% 0.5% 0 0 Asymptomatic Southeast Asian, Mediterranean, Indian, Middle Easten, African
α-thalassemia trait (minor)
(2/4 genes affected)
95 - 98% 1.5 - 3.0% 0.5% 0 0 Causes low MCV
Possible mild anemia
Typically asymptomatic
Southeast Asian, Mediterranean, Indian, Middle Easten, African
α-thalassemia intermedia (α+)
(3/4 genes affected)
60 - 90% < 2% < 1% 0.8 - 40% 2 - 5% Varies by severity
See alpha thalassemia
Southeast Asian, Mediterranean, Indian, Middle Easten, African
α-thalassemia major (αO)
(4/4 genes affected)
0 0 0 0 85 - 90% Typically fatal in utero (hydrops fetalis) Very rare in the U.S. (< 20 cases/year)
Beta (β) Thalassemias
Condition Hg A Hg A2 Hg F Hg S/C/H/Bart Clinical Affected populations
β-thalassemia trait (minor)
(1/2 genes affected)
92 - 95% > 3.5% 0.5 - 4% 0 Typically asymptomatic Mediterranean, Middle Easten, African, Central Asia, Indian, Far East
β-thalassemia intermedia (β+)
(2/2 genes affected)
10 - 30% 2 - 5% 70 - 90% 0 Varies depending on severity
See beta thalassemia
Mediterranean, Middle Easten, African, Central Asia, Indian, Far East
β-thalassemia major (βO)
(2/2 genes severely affected)
0 2 - 5% 95 - 98% 0 Severe
See beta thalassemia
Mediterranean, Middle Easten, African, Central Asia, Indian, Far East
Hemoglobin C disease
Condition Hg A Hg A2 Hg F Hg S Hg C Clinical U.S. prevalence
Hg C trait
(Hg A + Hg C)
55 - 65% < 3.5% < 1% 0 30 - 40% Typically asymptomatic 1 - 3% among blacks
Hg C disease
(Hg C + Hg C)
0 0 > 0.5% 0 > 90% Typically asymptomatic
See Hg C disease
< 0.10% among blacks
Sickle cell disease
Condition Hg A Hg A2 Hg F Hg S Hg C Clinical U.S. prevalence
Sickle cell trait
(Hb S + Hb A)
55 - 65% 1.5 - 3.0% 0.5% 35 - 45% 0 Typically asymptomatic
See sickle cell trait
8% among blacks
Sickle cell disease
(Hb S + Hb S)
0 1.5 - 3.0% 5 - 15% 80 - 95% 0 Severe
See sickle cell disease
0.3% among blacks
Hg S +
β thalassemia major (βO)
0 > 3.5% 5 - 15% 80 - 90% 0 Similar to sickle cell disease 1 - 3% among patients with sickle cell disease
Hg S + Hg C 0 < 3.5% < 3% 50 - 55% 40 - 45 Midler than sickle cell disease 25 - 30% among patients with sickle cell disease
Hg S +
β thalassemia intermedia (β+)
10 - 25% > 3.5% < 3% 70 - 80% 0 Midler than sickle cell disease 5 - 10% among patients with sickle cell disease


  • Reference [10,11,15]
Alpha thalassemia characteristics
α-thalassemia carrier
(1/4 genes affected)
  • Asymptomatic
  • RBC indices are normal or may have mild reduction in MCV (81±7) and MCH (26±2.3)
  • Hemoglobin pattern is normal
α-thalassemia trait (minor)
(2/4 genes affected)
  • Asymptomatic
  • RBC indices show reduction in MCV (71±4) and MCH (23±1.3)
  • Mild anemia may be present. Hemoglobin pattern is normal.
  • Often confused with iron deficiency anemia. RDW is typically elevated in iron-deficiency anemia and normal or close to normal in α-thalassemia trait.
α-thalassemia intermedia (α+)
(3/4 genes affected)
  • Clinical course varies depending on the amount of hemoglobin H produced. Most patients are clinically well and do not need transfusions.
  • Occasional transfusions may be necessary during hemolytic or aplastic crises in some patients
  • Disease requiring chronic transfusions is rare
α-thalassemia major (αO)
(4/4 genes affected)
  • Typically fatal in utero (hydrops fetalis). Fetuses are delivered stillborn at 30 - 40 weeks gestation or die soon after birth.
  • Ultrasound features include generalized edema and pleural and pericardial effusions as a result of congestive heart failure induced by severe anemia
  • Intrauterine transfusions and transfusions after birth may be attempted


  • Reference [10,16]
Beta thalassemia characteristics
β-thalassemia trait (minor)
(1/2 genes affected)
  • Typically asymptomatic. Mild anemia may be present.
  • RBC indices show a reduction in MCV (< 79) and MCH (< 27)
β-thalassemia intermedia (β+)
(2/2 genes affected)
  • Clinical course varies depending on the severity of anemia
  • Some patients may require chronic transfusions while others may not
β-thalassemia major (βO)
(2/2 genes severely affected)
  • Lifelong transfusions
  • Extramedullary hematopoiesis in the liver, spleen, and bones
  • Iron overload requiring chelation therapy
  • RBC indices show a severe reduction in MCV (50 - 70) and MCH (12 - 20)









  • References [12,13]
End-organ damage in sickle cell disease
Organ Comments
Blood and lymphatic system
  • Functional asplenia, chronic hemolysis and anemia, acute splenic sequestration, transient aplastic crises
Bones
  • Avascular necrosis of the shoulder, hip, and knee
Brain
  • Strokes, particularly in childhood. See stroke above.
  • Venous sinus thrombosis
  • Cognitive deficits
Cardiopulmonary
  • Acute chest syndrome, pulmonary hypertension, restrictive cardiomyopathy, tricuspid regurgitation, restrictive lung disease, arrhythmia and sudden death
  • Cardiopulmonary complications accounted for 45% of deaths in one study of sickle cell patients
Eyes
  • Proliferative retinopathy
Gastrointestinal
  • Cholelithiasis, bile duct disease, hepatic venous congestion, mesenteric infarction
Skin
  • Avascular necrosis leading to leg ulcers
Urogenital
  • Glomerulosclerosis, proteinuria, hematuria, kidney failure, priapism







  • Reference [9]
RISK FACTORS FOR IRON-DEFICIENCY ANEMIA
Pathology Risk factors
Increased demand
  • Childhood growth
  • Pregnancy (2nd and 3rd trimester)
  • Use of erythropoietin agents (e.g. Epogen®) in chronic kidney disease
Insufficient intake
  • Vegetarian or vegan diet - heme iron in meat is absorbed much more readily than nonheme iron found in fruits, vegetables, and iron-fortified foods
  • Infants who consume excessive cow's milk
Impaired absorption
  • Gastric and/or duodenal resection
  • Bariatric surgery
  • Celiac disease
  • H. pylori infection
  • Atrophic gastritis
  • Crohn's disease
  • Proton pump inhibitors (e.g. Nexium®) and H-2 blockers (e.g. Pepcid®)
  • Tannins (found in tea)
  • Soy protein
  • Calcium
  • Polyphenols
  • Phytates (found in legumes and whole grains)
  • Intestinal helminth infections (e.g. hookworm)
  • Iron-refractory iron-deficiency anemia - rare genetic condition that leads to chronically elevated hepcidin levels
Blood loss
  • Menses
  • Blood donation
  • Gastrointestinal bleeding (cancer, inflammatory bowel disease, ulcers, angiodysplasia, etc.)
  • Hereditary hemorrhagic telangiectasia - rare genetic condition where arteriovenous malformations (AVMs) form that are prone to hemorrhage
  • Intestinal helminth infections (schistosomiasis)
Chronic hemolysis
  • Paroxysmal nocturnal hemoglobinuria
  • Cold agglutinin disease
  • Autoimmune hemolytic anemia
  • Mechanical heart valves








  • 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









  • 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





  • 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


  • 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.


  • 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
  • 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.








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 typically cause RBC agglutination.

Cold autoimmune hemolytic anemia (CAHA)
  • AHA is called "cold" because binding is maximal at room temperature down to 40° F. CAHA is less common than WAHA accounting for 15 - 25% of AHA cases. CAHA is caused by IgM antibodies that coat RBCs and fix complement (C3) to the cell. If the complement system is completed, the cell may undergo intravascular hemolysis, but in most cases, the C3-tagged cell is destroyed in the liver or spleen by macrophages. IgM antibodies can also cause RBCs to agglutinate and this is why they are sometimes referred to as "cold agglutinins." Patients with cold agglutinins may develop acrocyanosis (bluish or purple toes or fingers) when exposed to colder temperatures.

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, myelodysplasia, 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
  • Artificial heart valves are thought to damage RBCs through increased shear stress. Symptomatic hemolysis is rare occurring in < 1% of patients with modern 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. Paravalvular leak is thought to be the main mechanism behind valve-related hemolysis. [42]

Thrombotic thrombocytopenic purpura (TTP)
  • Incidence - Overall, TTP is a very rare condition with an incidence of 2.9 per 1 million/year in adults and 0.1 per 1 million/year in children.
  • Pathology - TTP is caused by a deficiency in an enzyme called ADAMTS13 that is responsible for cleaving von Willebrand factor multimers that are secreted from vascular endothelial cells. Deficiencies can be inherited through abnormal ADAMTS13 genes or they can be acquired through the development of anti-ADAMTS13 antibodies (more common). TTP causes clots to form in small blood vessels, and when RBCs travel through these damaged vessels, they are hemolyzed.
  • Risk factors - Risk factors for acquired TTP include pregnancy, cancer, HIV, lupus, infections, chemotherapy, ticlopidine, clopidogrel, cyclosporine, and quinine.
  • Treatment - 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. Immunosuppressants such as corticosteroids are also frequently given. [43]

Hemolytic uremic syndrome (HUS)
  • Pathology - HUS is a condition that occurs when the endothelial cells in small blood vessels become damaged and thrombi form. RBCs traveling through the damaged and thrombosed vessels are hemolyzed, and the microcirculation in the kidneys is occluded leading to renal 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 cases) is caused by a toxin that is released by bacteria, primarily Shigella dysenteriae and E. Coli. Stx-HUS patients will present with severe abdominal pain and diarrhea that is often bloody. Stx-HUS is most common in children < 5 years of age with an incidence of 6.1 cases per 100,000/year. Treatment of Stx-associated HUS is generally supportive and most patients make a full recovery.
  • Atypical HUS - Atypical HUS (5 - 10% of cases) is not as completely understood as Stx-HUS. Endothelial damage in atypical HUS is thought to occur through uncontrolled activation of the complement pathway. Risk factors for atypical HUS include Streptococcus pneumoniae infection, viruses, drugs, cancer, transplantation, and inherited forms. Atypical HUS has an incidence of about 2 cases per 100,000/year in the general population. Atypical HUS can be treated with drugs that inhibit the complement system (eculizumab and ravulizumab) and plasma exchange. [43,44]

Disseminated intravascular coagulation (DIC)
  • Pathology - DIC is a condition where the coagulation pathways are inappropriately activated leading to the consumption of coagulation factors, microvascular thrombosis, hemorrhaging, and multiple organ failure. RBCs traveling through the 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, and trauma.
  • Treatment - 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 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 makes RBCs much more susceptible to complement activation and hemolysis. The name 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. An anti-complement antibody called eculizumab (Soliris) was approved to treat the disease in 2007, and it has greatly improved mortality. [41]

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


  • 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. Cold agglutinins may form secondary to an underlying condition (e.g. cancer, Mycoplasma pneumonia, mononucleosis), or they may occur in the absence of an identifiable cause in which case a person is considered to have "cold agglutinin disease."
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, transfusion
  • VTE prophylaxis - all hospitalized patients should receive VTE prophylaxis; consider VTE prophylaxis in ambulatory patients at increased risk [38,40]
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. 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




  • References [48, 49, 53]
Megaloblastic causes
Vitamin B12 deficiency
Folate/folic acid deficiency (Vitamin B9)
  • Folate deficiency is typically functional or related to decreased absorption. Dietary deficiency is rare with the exception of alcoholics. Folate is naturally occurring and found in dark leafy green vegetables, citrus fruits, and liver. Folic acid is a synthetic compound found in supplements and fortified foods such as breads and cereals.
  • 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
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
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
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 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 (Imuran®)
  • Azathioprine
  • 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®)











  • 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%







  • Reference [6]
Iron form % elemental iron Other
Ferrous gluconate 12%
  • 325 mg tablet = 39 mg elemental iron
  • Available over-the-counter
Ferrous sulfate 20%
  • 325 mg tablet = 65 mg elemental iron
  • Fer-In-Sol® drops (75 mg/ml) = 15 mg elemental iron/ml
  • Available over-the-counter
Ferrous fumarate 33%
  • 325 mg tablet = 107 mg elemental iron
  • Ferrous fumarate is available over-the-counter and it is also found in birth control pills





  • * 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


  • 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


  • 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






  • References [9]
Iron study values in certain conditions
Condition Ferritin Iron level Transferrin saturation TIBC
Iron-deficiency anemia Low Low Low High
Anemia of chronic disease > 100 Low Low / Normal Low
Anemia of chronic disease and Iron-deficiency anemia < 100 Low Low / Normal Low
Hemochromatosis High High High Low
Hemolytic anemia High High High Normal / Low
Sideroblastic anemia High Normal / High High Normal / Low
Iron poisoning Normal High High Normal